**2.2. Effects of supplements with Sea-buckthorn fractions on dyslipidemia associated to childhood obesity**

Sea-buckthorn (Hippophae rhamnoides) is a plant of seashores and cliffs; it can be found in Central Asia and Europe, all the way from the shores of the Black Sea to the northwestern shores of the continent. In addition, Sea-buckthorn has spread to Canada and the United States and nowadays the plant has been currently domesticated. Both the berries and the leaves of the plant can be used as dietary supplements.

According to Greek legend, the sick and wounded horses of warriors would be allowed to roam and feed on the shrub. The horses would return more strong and healthy. Sea-buckthorn is a special food that is used to help astronauts maintain their health in space [55].

Since ancient times, Sea-buckthorn berries have been used for their beneficial effects on blood circulation, skin regeneration, and anti-inflammatory effects. The berry contains oil, both in the seed and in the soft parts (pulp oil from the flesh and peel). Special features of the oils are high proportions of palmitic (16:0), oleic (18:1n-9), palmitoleic (16:1n-7), linoleic (18:2n-6), and α-linolenic (18:3n-3) acids as well as vitamin E, carotenoids, and sterols [56].

While the seed oil is rich in linoleic and α-linolenic acids, the pulp oil is a very rich source of palmitoleic acid. This acid is an essential fatty acid that is rare to find in the natural form. Macadamia nuts are the only other source of Omega-7 fatty acids and only in trace amounts [57].

The pulp oil provides a 1:1 ratio of Omega-3/Omega-6. The pulp can be consumed either as a juice or a puree. All the components of the berry contain flavonoides, but the seed residues and the leaves are the most important sources of flavonoides (particularly quercetin, isorham‐ netin, kaempherol) [58]. Flavonoides and vitamin C have anti-inflammatory effects and act through a synergic mechanism [59].

The Sea-buckthorn fruit is a very rich source of vitamin C (695 mg per 100 grams), about 12 times greater than oranges, placing Sea-buckthorn fruit among the most enriched plant sources of vitamin C [60].

The origin (subspecies of more than seven) and harvesting time of the berries, as well as oil isolation technology, influence the oil composition [61]. The amounts of bioactive compounds in sea buckthorn berry vary depending on the subspecies, area and year of cultivation, and the maturity of the berries [62].

The method used for extracting Sea-buckthorn influences the potency and the quality of the oil. Cold pressing, hot pressing, solvent extraction, and maceration in other carrier oils are the most used ways for Sea-buckthorn oil extraction, but each method have its own disadvantages. For example, cold-pressing may be ideal, but the extraction rate is quite low. Hot-pressing destroys healthy nutrients and solvent extraction may contaminate the oil with hazardous solvents. The best way to obtain Sea-buckthorn oil is through supercritical CO2-extraction.

Potentially all compounds of the berry, including flavonols, carotenoids, fatty acids, toco‐ pherols, tocotrienos, and phytosterols of the pulp oil, can affect the metabolic profile. Clinical and experimental studies have demonstrated a wide range of positive effects of the sea buckthorn oils and flavonoides on the lipid profile. Animal and in vitro studies have suggested that sea buckthorn oils [63, 64, 65] and alcohol extracts and flavonoid preparations [66, 67, 68, 69] have antioxidant and anti-inflammatory activities and may beneficially affect serum glucose and triglyceride concentrations. The effect was observed in participants who had a metabolic profile that reflected higher cardiometabolic risk.

In two clinical studies [70, 71] involving healthy men, the intake for 8 weeks of Seabuckthorn juice versus placebo or supplementation with 5 g of Sea-buckthorn berry oil for 4 weeks versus coconut oil control had no significant influence on the lipid profile (total cholesterol, LDL-c, HDL-c, and triglycerides), but decreased moderately the susceptibility of LDL to oxidation [71].

In healthy, normolipidemic adults having healthy diets, consumption of Sea-buckthorn berry did not affect the circulating concentration of lipid markers, but increased the fasting plasma concentration of quercetin and isorhamnetin indicating that Sea-buckthorn berry is a good dietary source of flavonols [72].

There is a paucity of clinical and animal experiments focused on Sea-buckthorn oil effects on dyslipidemia associated to obesity and the studies are lacking in childhood obesity. Also, in some published data, the tested oils are not defined and some studies should have a better design. The influence of the oils on the plasma lipid levels needs further investigation.

In the experimental study done on white albino rabbits fed with high cholesterol, CO2 extracted Sea-buckthorn seed oil treatment (1 ml for 30 days) had significant anti-atherogenic and cardioprotective activity. The treatment increased the HDL-c plasma levels and restored the acetylcholine-induced vasorelaxant effect to that of normal values [73].

A randomized, double-blind, crossover study including two 4-week periods with either 3 g/day of black currant seed oil or 2.8 g/day of fish oil separated by a 4-week washout period was done on 15 healthy females. The results showed that black currant seed oil had minor changes on serum n3 fatty acids. Serum levels of LDL cholesterol were lower (p < 0.05) after black currant seed oil compared to fish oil. Plasma glucose concentration decreased during the fish oil supplementation (p < 0.05). The results underline the beneficial effects of berries and berry seed oils on serum lipid profile [74].

The origin (subspecies of more than seven) and harvesting time of the berries, as well as oil isolation technology, influence the oil composition [61]. The amounts of bioactive compounds in sea buckthorn berry vary depending on the subspecies, area and year of cultivation, and the

The method used for extracting Sea-buckthorn influences the potency and the quality of the oil. Cold pressing, hot pressing, solvent extraction, and maceration in other carrier oils are the most used ways for Sea-buckthorn oil extraction, but each method have its own disadvantages. For example, cold-pressing may be ideal, but the extraction rate is quite low. Hot-pressing destroys healthy nutrients and solvent extraction may contaminate the oil with hazardous solvents. The best way to obtain Sea-buckthorn oil is through supercritical CO2-extraction.

Potentially all compounds of the berry, including flavonols, carotenoids, fatty acids, toco‐ pherols, tocotrienos, and phytosterols of the pulp oil, can affect the metabolic profile. Clinical and experimental studies have demonstrated a wide range of positive effects of the sea buckthorn oils and flavonoides on the lipid profile. Animal and in vitro studies have suggested that sea buckthorn oils [63, 64, 65] and alcohol extracts and flavonoid preparations [66, 67, 68, 69] have antioxidant and anti-inflammatory activities and may beneficially affect serum glucose and triglyceride concentrations. The effect was observed in participants who had a

In two clinical studies [70, 71] involving healthy men, the intake for 8 weeks of Seabuckthorn juice versus placebo or supplementation with 5 g of Sea-buckthorn berry oil for 4 weeks versus coconut oil control had no significant influence on the lipid profile (total cholesterol, LDL-c, HDL-c, and triglycerides), but decreased moderately the susceptibility

In healthy, normolipidemic adults having healthy diets, consumption of Sea-buckthorn berry did not affect the circulating concentration of lipid markers, but increased the fasting plasma concentration of quercetin and isorhamnetin indicating that Sea-buckthorn berry is a good

There is a paucity of clinical and animal experiments focused on Sea-buckthorn oil effects on dyslipidemia associated to obesity and the studies are lacking in childhood obesity. Also, in some published data, the tested oils are not defined and some studies should have a better design. The influence of the oils on the plasma lipid levels needs further investigation.

In the experimental study done on white albino rabbits fed with high cholesterol, CO2 extracted Sea-buckthorn seed oil treatment (1 ml for 30 days) had significant anti-atherogenic and cardioprotective activity. The treatment increased the HDL-c plasma levels and restored the

A randomized, double-blind, crossover study including two 4-week periods with either 3 g/day of black currant seed oil or 2.8 g/day of fish oil separated by a 4-week washout period was done on 15 healthy females. The results showed that black currant seed oil had minor changes on serum n3 fatty acids. Serum levels of LDL cholesterol were lower (p < 0.05) after black currant seed oil compared to fish oil. Plasma glucose concentration decreased during the

acetylcholine-induced vasorelaxant effect to that of normal values [73].

metabolic profile that reflected higher cardiometabolic risk.

maturity of the berries [62].

66 Lipoproteins - From Bench to Bedside

of LDL to oxidation [71].

dietary source of flavonols [72].

In a study group including 49 atopic dermatitis patients who took 5 g (10 capsules) of seed oil, pulp oil, or paraffin oil daily for 4 months, a significant (p < 0.05) increase in the level of high-density lipoprotein cholesterol from 1.38 to 1.53 mmol/L was observed in the pulp oil group [75].

It was demonstrated that a high-MUFA, cholesterol-lowering diet may be superior to a lowfat diet because it lowers triglyceridemia and does not decrease the plasma level of HDL cholesterol [76].

C16:1n7-palmitoleate is known as an adipose tissue-derived lipid hormone that works at the crosstalk between lipid and sugar metabolism. The acid stimulates muscle insulin action and suppresses hepatosteatosis [77]. The following lines will present the effects of palmitoleic acid on insulinodependent tissues.

In vitro studies done on rat L6 skeletal muscle cells, it was demonstrated that palmitoleic might facilitate uptake and utilization of glucose by upregulation of the activities of the glucose transporters GLUT1 and GLUT4 [78].

On a spontaneous model of obese type 2 diabetes rats, researchers demonstrated that repeated administration of palmitoleic acid increased insulin sensitivity by down-regulating mRNA expressions of proinflammatory adipocytokine genes (TNFα and resistin) in white adipose tissue and by decreasing hepatic lipid accumulation. Palmitoleic acid down-regulates the mRNA of lipogenic genes (as an example, SREBP-1) in the liver and reduces both the plasma triglyceride and hepatic triglyceride levels [79].

It is worth mentioning the effects of C16, n-7 on pancreatic tissue. It was demonstrated that palmitoleic and oleic acids prevent the deleterious effects of saturated fatty acids and high glucose on human pancreatic beta-cell turnover and function via Bcl-2 [80].

In a recent randomized crossover study, 80 overweight women were divided into four groups with different supplements intake for 30 days: dried Sea-buckthorn berries, Sea-buckthorn oil, Sea-buckthorn phenolics ethanol extract mixed with maltodextrin (1:1), or frozen bilberries. Sea-buckthorn-induced effects were mainly on serum triglycerides and very-low-density lipoprotein (VLDL) and its subclasses in the groups with higher metabolic risk. From the supplements, Sea-buckthorn oil induced a decreasing trend in serum total, IDL, and LDL cholesterol and apolipoprotein B in participants with the baseline metabolome of higher cardiometabolic risk [81].

In the Sea-buckthorn, most antioxidants appear to accumulate in the seeds relative to the pulp, leaves, or stem despite most flavonoids being in the leaves. The total phenolic content of the leaves is high and is between 47.06–66.03 mg/g rutin equivalents (RE) [82].

Phenolic compounds and flavonoids ameliorate bodyweight, blood glucose, and serum lipid profile. Also flavonoids from seeds and leaves have anti-obesity and hypoglycemic effect.

Clinical studies have demonstrated that the berry and the ethanol fraction from Sea-buckthorn pulp has beneficial effects on postprandial glucose and insulin levels [83].

Experimental studies done in diabetic rats demonstrated that flavonoids present in water extracts of Sea-buckthorn seeds have hypoglycemic and triglyceride-lowering effect [84].

The fibers and polyphenols in Sea-buckthorn (Hippophaë rhamnoides) extraction residues delay also the postprandial lipemia [85].

Quercetin, as an important flavonoid in the Sea-buckthorn, has also hypolipidemic effect [86].

Quercetin inhibits fatty acid and triacylglycerol synthesis in rat liver cells [87].

In vitro studies demonstrated that quercetin and isorhamnetin have protective effects against oxidized LDL-induced endothelial cell injuries. The flavonoids' beneficial effects might derive from their antioxidant activity and from their capability in modulating the expression of eNOS (endothelial nitric oxide synthase) and LOX-1(lipooxygenase-1) [66].

There are some molecules that are (currently known to be) unique to Sea-buckthorn named flavonoid glycosides [88] and they have antioxidant activity.

High-fat diet obese C57BL/6J mice treated with 70% ethanolic extract of Sea-buckthorn at 500– 1,000 mg/kg bodyweight over 13 weeks had lower hepatic and serum total cholesterol, lower hepatic triglycerides and serum leptin level versus non-treated mice. Triglyceridemia and insulinemia were similar in the studied group. The study demonstrated that the Sea-buckthorn intervention was effective in preventing body weight gain and fat accumulation in the liver. The molecular mechanism of this effect is the increase of the hepatic mRNA expression of peroxisome proliferator-activated receptor (PPAR) α and PPAR-γ while the level of the hepatic key enzyme in the fatty acid synthase was decreased [89].

Many compounds from Sea-buckthorn help lose extra weight. Omega-7 in Sea-buckthorn sends messages to the brain, telling it to stop storing calories as excess fat. Sea-buckthorn oil stimulates healthy bowel moves, thereby protecting cell membranes from oxidative and physical stress.

In overweight or obese women, the intake of different berries and berry fractions for 33 days with washout periods of 30–39 days have resulted in positive effects such as a significantly decrease in the waist circumference and in the level of vascular cell adhesion and intercellular adhesion molecules [90].

## **2.3. Association of the atherogenic indexes with antropometric, inflammatory, oxidative stress and endothelial dysfunctional markers in childhood obesity**

It was demonstrated that in obese children with metabolic syndrome, there is a positive association between the high waist circumference and the atherogenic index (total cholesterol/ HDL-c) [91].

Endothelial dysfunction, inflammation, and oxidative stress are present in childhood obesity. Especially during puberty, there are some pro-inflammatory and pro-oxidative changes associated with a relative insulin resistance. The association of the inflammatory and oxidative stress markers with the high value of the apo B/apo A1 ratio in obese children underlies the action in the cluster of different pathogenic mechanisms, augmenting the atherosclerosis development [26, 92].

Apolipoproteins B and A-1 are proposed as markers with value in pediatric lipid risk assess‐ ment. High apo B and low apo A-1 levels, usually present in obese children and adolescents reflect a lipoprotein profile predisposing to the development of subclinical atherosclerosis in adulthood [93], [94].

The concentrations of plasma apolipoprotein (apo) B are often increased in childhood obesity, partly due to the hepatic overproduction of apo B containing lipoproteins [95,96].

Many researchers consider apo B as a better predictor of vascular risk than LDL because apo B, in comparison with LDL, is more strongly associated with other cardiovascular risk factors [97].
