**9. Health benefits of SB berry polyphenols (in powders, purees, and extracts)**

## **9.1 Anti-inflammatory effect**

Two grams of frozen SBB puree containing 16.7 mg of flavanol glycosides or a placebo was consumed for 3 months in a study including 254 healthy volunteers [41]. The objective was to assess the efficacy and safety of SBB for common cold (CC), digestive tract infections (DTI), and urinary tract infections (UTI). While no difference was reported in CC and DTI frequency or duration, consumption of SBB reduced both the number and duration of UTI. A small but significant decrease in CRP was also observed in the SBB group. The decrease in the inflammatory marker CRP was confirmed in another study in which a dose of 28 g of SBB or placebo was given to 220 healthy volunteers for 90 days. SBB did not affect serum total HDL and LDL cholesterol, nor serum triacylglycerol concentrations. However, compared with placebo, there was a significant reduction in blood concentrations of CRP in the SBB group [9].

#### **9.2 Regenerative effect for improvement of pancreatic function**

SBB pulp at a daily dose of 1 or 2 ml/kg bw for 3 weeks was administered to streptozotocin-nicotinamide (STZ) induced diabetes in rats [42]. A decrease of more than 50% of fasting hyperglycemia was observed in diabetic rats, at both 1 and 2 ml/ kg. Pancreatic glutathione content increased significantly in SBB treated diabetic rats Moreover, a decrease in HbA1c was reported at the highest dose. SBB decreased all histopathological changes induced by STZ, such as degenerative and lytic changes were reduced, beta cells depletion was decreased, as well as fibrosis. SBB pulp had a regenerative and protective effect on pancreatic beta cells.

#### **9.3 Metabolic health improvement**

The beneficial effect of flavonoid-rich extract of SBB was assessed in high-fat diet–induced obesity (HFDO) at daily doses of 100 and 300 mg/kg bw for 9 weeks, and compared to placebo [43]. SBB administration significantly reduced body weight gain, inhibited macrophage infiltration into adipose tissues, and downregulated TNFα mRNA expression in adipose tissue. A decrease in TG was observed but not in total cholesterol. At the highest dose of 300 mg/kg, hepatic TG was decreased by 49.56% when compared to HFDO control mice. Blood glucose concentration was 14.55% lower in the SBB treatment group (300 mg/kg), compared to the HFDO control. SBB alleviated the glucose intolerance induced by HFD, as determined by Oral Glucose Tolerance Test (OGTT). The sizes of adipocytes were considerably lower at both doses of SBB, compared to the HFDO control. Therefore, the anti-obesity activity of SBB may be attributed partly to a decrease in the volume of fat cells. Decrease in adipose tissue inflammation, anti-obesity properties, improvement of glucose tolerance and glycemia, and the decrease in hepatic TG accumulation all points to an improvement of cardiometabolic profile.

The efficacy of SBB was also evaluated in a model of spontaneously hypertensive stroke-prone rat, using a daily dose of 0.7 g/kg-bw for 2 months [44]. Mean and diastolic blood pressure, heart rate, total plasma cholesterol, triglycerides, and glycated hemoglobin were significantly decreased by the SBB treatment when compared to hypertensive control group. The number of AP-containing capillary portions fell

while the number of those containing DPPIV increased. The expression of these 2 enzymes is modulated by inflammation enhanced by hypertension. Antihypertensive and cardioprotective properties measured by heart rate, blood pressure, total plasma cholesterol, TG levels improvement were thus confirmed.

The efficacy of SBB, and SBB phenolic extract on metabolic health was compared to bilberry berries (BB) in 110 overweight women [45]. The daily doses were all equivalent to 100 g of fresh berries. Each product was consumed for 35 days in a cross-over study. Decrease in waist circumference and TNFα, and a small decrease in fasting plasma glucose was observed after SBB consumption. A decrease in ICAM and TNFα was observed after consumption of SB extract. No significant difference in BP, percentage of fat mass, fasting plasma cholesterol, TG, and IL6 levels was observed. Therefore, SB products brought mild but significant improvement in metabolic, inflammatory and endothelial markers in overweight volunteers. Another clinical study demonstrated the efficacy of SB juice (SBJ) on platelet aggregation. Placebo or 300 ml of SBJ was consumed by 20 healthy volunteers for 8 weeks [46]. No difference in platelet aggregation and LDLox levels was seen between placebo and SBJ group. A non-significant increase of 20% was observed in plasma HDL-C. In another study, a crude flavone extract from SBB prevented thrombogenesis in an *in vivo* model of thrombosis in mouse femoral artery, probably by inhibition of platelet aggregation. It prolonged occlusion time at a dose of 300 μg/kg had a similar effect to aspirin at 10 mg/kg [47].

This cardioprotective effect and improvement in metabolic profile has been confirmed in clinical studies. Eighty overweight women were given either 20 g of dried SBB, or 14.6 g of sea buckthorn phenolic extract for 35 days, in a randomized cross-over study. All these daily doses represent 100 g of fresh SBB [16]. All groups using the various SB products showed significant improvement in metabolic profile, especially in individuals with higher baseline cardiometabolic risk. SB-induced effects were mainly on serum TG and very-low-density lipoprotein (VLDL) and its subclasses, which decreased in participants with higher baseline cardiometabolic risk. During SB consumption, a significant decrease in TG and creatinine was observed. To conclude, a meta-analysis including 11 RCT which enrolled 514 patients confirmed that supplementation with SBB and extracts significantly reduced total cholesterol, LDL-cholesterol and significantly increased HDL-cholesterol in subjects with cardiovascular risks [6].

#### **9.4 Hepatoprotective effect**

Sea buckthorn extract (SBE) was administered at the daily dose of 15 g, 3 times a day for 6 months, to 50 cirrhotic Child-Pugh grade A and B patients [48]. The rate of normalization in AST and ALT, was significantly higher in the groups treated with SBE: 80% in the treated group and 56% in the control group. Parameters of liver fibrosis such as serum laminin, hyaluronic acid, total bile acid (TBA), collagen types III and IV were decreased after treatment, when compared with control group. SBE decreased markers of liver fibrosis and improved the rate of AST/ALT normalization, suggesting hepatoprotective properties.

#### **9.5 Regenerative effect**

Stemberry®, a SBB aqueous extract standardized in 30% of proanthocyanidins was consumed by 12 healthy participants at a daily dose of 500 mg, compared to a placebo in one single dose. Rapid and highly selective stem cell mobilization was observed, as quantified by an increase in the number of circulating CD45dim C D34<sup>+</sup> CD309<sup>−</sup> progenitor stem cells by 24%, CD45− CD31+ CD309+ endothelial stem cells by 33%, and CD45− CD90+ mesenchymal stem cells by 20%. All these types of stem cells are involved in regenerative and reparative functions. Moreover, a mild significant increase was observed in the number of CD45dim CD34+ CD309+ pluripotential stem cells [49]. SB PAC-rich extract supports the natural ability of the body to repair and renew, suggesting regenerative properties.

#### **9.6 Acute lung injury protection**

The efficacy of SBB paste (SB total polyphenols 191.5 mg/g and SB total flavonoids 130.9 mg/g) was studied in a mouse model of LPS-induced acute lung injury [50]. SBB paste was consumed for 7 days at daily doses of 200, 400 and 800 mg/kg bw, and at day 8 LPS challenge was carried out. The loss of body weight, microstructure lesions in the lung tissue, increase in MDA, and reduction of SOD and glutathione peroxidase levels caused by LPS challenge were all significantly reduced by SB treatment in a dose dependent manner. As a consequence, SBB paste improved lung lesions such as alveolar thickness caused by edema, hemorrhage alveolus collapse, inflammatory cell inflammation were greatly reduced in the SB-treated group compared with the group acute lung injury. SB treatment provided significant protection against protein transvascular leakage. As lung lesions, oxidative stress markers are decreased, SB provides protection against acute lung injury, via partly the activation of Nrf2 pathway and redox homeostasis due its high content in polyphenols.

#### **9.7 Cytoprotective and antioxidant effect**

SBB flavones at the concentration of 100 ug/mL exert cytoprotective and antioxidant properties in a tert-Butyl hydroperoxide-induced cytotoxicity (BOOH) model in lymphocytes [51]. SBB flavones significantly inhibited tert-BOOH-induced cytotoxicity and free radical production, restored the antioxidant status, significantly maintained ATP levels comparable to control and protected the cells from tert-BOOH-induced lipid peroxidation. Treatment with SBB flavones reduced tert-BOOHinduced apoptosis and a decreased tert-BOOH-induced formation of DNA breaks by 30%. Cytoprotective and antioxidant effects suggest safety of SB berries extracts.

#### **9.8 Anti-inflammatory and neuroprotective effect**

The efficacy of a SBB extract rich in flavonoids was demonstrated in a model of high-fat high-fructose diet (HFFD) induced cognitive impairment [11]. The extract was consumed for 14 days at 2 daily doses of 100 and 500 mg/kg-bw. Compared to HFFD placebo mice, SBB consumption resulted in a reduction in body weight gain by 8.8% and a decrease in glucose intolerance. It also improves insulin sensitivity. More specifically, SBB consumption resulted in a 45–48% decrease in HOMA-IR value, a 20–30% decrease in fasting hyperglycemia, a 12–20% decrease in fasting insulinemia, a reduction in TG and total cholesterol levels, a prevention of neuronal loss and working memory impairment in behavioral tests, and a suppression of HFFD-induced synaptic dysfunction and neuronal damages. Dietary supplementation SF significantly improved the length by 37.91% and width by 10.07% of postsynaptic density in the hippocampus when compared with the HFFD group mice. SBB flavonoids also increased the levels of BDNF, NT-3, NT-4 and NGF involved in the growth, survival, and synaptic plasticity of brain neurons. SBB flavonoids also reversed HFD-induced

overexpression of iNOS, and the up-regulation of p38 phosphorylation and NFkB expression, which are markers of neuro-inflammation.

As a consequence, SBB flavonoids displayed neuroprotective effects against chronic neuro and systemic inflammation observed in diabetes-induced obesity and is associated with an improvement of metabolic parameters (namely lipid and glucose profiles).

## **9.9 Beneficial effect in idiopathic nephrotic syndrome**

Hydroalcoholic extract of leaves and fruits of SB at a daily dose of 350 mg twice daily for 12 weeks, in addition to other standard drugs, was administered to 52 patients with Idiopathic Nephrotic Syndrome [52]. Beneficial effects were reported in symptoms like anorexia and oedema. There was no statistically significant change in creatinine, phosphorous and blood urea after 12 weeks of treatment when compared to control subjects. Improvement in cholesterol, triglyceride, albumin and 24-hour urinary protein excretion in the SB group was observed. Decreased CRP and IL6 levels were also noticed in the group treated with SB, confirming a nephroprotective role of SB.
