Black Raspberry Extract Increased Circulating Endothelial Progenitor Cells and Improved Arterial Stiffness in Patients with Metabolic Syndrome: A Randomized Controlled Trial

Not to be followed unless your doctor prescribes this. You know how dangerous this stuff can be.

Interesting that no changes in blood pressure were seen  but other markers were better that your doctor can explain to you.

Black Raspberry Extract Increased Circulating Endothelial Progenitor Cells and Improved Arterial Stiffness in Patients with Metabolic Syndrome: A Randomized Controlled Trial

To cite this article:
Jeong Han Saem, Kim Sohyeon, Hong Soon Jun, Choi Seung Cheol, Choi Ji-Hyun, Kim Jong-Ho, Park Chi-Yeon, Cho Jae Young, Lee Tae-Bum, Kwon Ji-Wung, Joo Hyung Joon, Park Jae Hyoung, Yu Cheol Woong, and Lim Do-Sun. Journal of Medicinal Food. April 2016, 19(4): 346-352. doi:10.1089/jmf.2015.3563.

Published in Volume: 19 Issue 4: April 13, 2016
Online Ahead of Print: February 18, 2016

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Author information

Han Saem Jeong,^1,* Sohyeon Kim,^1,* Soon Jun Hong,¹ Seung Cheol Choi,¹ Ji-Hyun Choi,¹ Jong-Ho Kim,¹ Chi-Yeon Park,¹ Jae Young Cho,¹ Tae-Bum Lee,² Ji-Wung Kwon,² Hyung Joon Joo,¹ Jae Hyoung Park,¹ Cheol Woong Yu,¹ and Do-Sun Lim¹

¹Department of Cardiology, Cardiovascular Center, Korea University Anam Hospital, Seoul, Korea.

²Gochang Black Raspberry Research Institute, Gochang, Korea.

^*These authors contributed equally to this work.

Address correspondence to: Soon Jun Hong, MD, PhD, Department of Cardiology, Cardiovascular Center, Korea University Anam Hospital, 126-1, 5ka, Anam-dong, Sungbuk-ku, Seoul 136-705, Republic of Korea, E-mail: psyche94@gmail.com

Manuscript received 22 July 2015
Revision accepted 14 January 2016

ABSTRACT

Administration of black raspberry (Rubus occidentalis) is known to improve vascular endothelial function in patients at a high risk for cardiovascular (CV) disease. We investigated short-term effects of black raspberry on circulating endothelial progenitor cells (EPCs) and arterial stiffness in patients with metabolic syndrome. Patients with metabolic syndrome (n = 51) were prospectively randomized into the black raspberry group (n = 26, 750 mg/day) and placebo group (n = 25) during the 12-week follow-up. Central blood pressure, augmentation index, and EPCs, such as CD34/KDR⁺, CD34/CD117⁺, and CD34/CD133⁺, were measured at baseline and at 12-week follow-up. Radial augmentation indexes were significantly decreased in the black raspberry group compared to the placebo group (−5% ± 10% vs. 3% ± 14%, P < .05). CD34/CD133⁺ cells at 12-week follow-up were significantly higher in the black raspberry group compared to the placebo group (19 ± 109/μL vs. −28 ± 57/μL, P < .05). Decreases from the baseline in interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were significantly greater in the black raspberry group compared to the placebo group (−0.5 ± 1.4 pg/mL vs. −0.1 ± 1.1 pg/mL, P < .05 and −5.4 ± 4.5 pg/mL vs. −0.8 ± 4.0 pg/mL, P < .05, respectively). Increases from the baseline in adiponectin levels (2.9 ± 2.1 μg/mL vs. −0.2 ± 2.5 μg/mL, P  < .05) were significant in the black raspberry group. The use of black raspberry significantly lowered the augmentation index and increased circulating EPCs, thereby improving CV risks in patients with metabolic syndrome during the 12-week follow-up.

Introduction

Black raspberry (Rubus occidentalis) has long been used in traditional alternative medicine in Korea because of its potential to improve vascular function. The components of black raspberry include flavonoids, tannins, phenolic acids, tyrosol, ellagitannins, and resveratrol.^1–4 These components have well-documented anti-inflammatory, antioxidative, and antiatherosclerotic effects.^5–7 Antioxidant compounds of black raspberry reduce blood pressure and improve arterial stiffness.⁸ Some studies have demonstrated that black raspberry improves blood pressure, lipid profiles, and vascular function.^9–11
Metabolic syndrome is a cluster of cardiovascular (CV) risk factors. As CV events are higher in patients with metabolic syndrome compared to the general population, it is important to manage individual CV risk parameters such as central blood pressure, radial artery augmentation index, and systemic inflammation.^12,13 Central blood pressure is more related to CV risk than peripheral blood pressure.¹⁴ Arterial stiffness is included in the European Society of Hypertension/European Society of Cardiology guidelines for the assessment of CV risk.¹⁵ Obesity, hypertension, dyslipidemia, and insulin resistance are all components of the metabolic syndrome and these factors are known to be associated with arterial stiffness.¹⁶ Arterial stiffness is not just an aging process, but a consequence of pathophysiological alterations of endothelial cells, vascular smooth muscle cells, and the extracellular matrix.^17,18 Especially, increased vascular stiffness is important in predicting CV disease in obese and insulin-resistant patients.¹⁹ The number of circulating endothelial progenitor cells (EPCs) promoting regeneration in damaged vascular endothelium and ischemic tissues is an emerging marker in CV diseases in recent years.²⁰ Inverse correlation between the number of circulating EPCs and CV risk factors has been reported.²¹ Oxidative stress results in downregulation of nitric oxide (NO), thereby leading to endothelial dysfunction, which promotes vasoconstriction, platelet activation, vascular inflammation, and arterial stiffness.²² Thus, the key clinical implication in managing metabolic syndrome is to block the subsequent complications of metabolic syndrome.

Management of metabolic syndrome encompasses lifestyle modification and lowering individual CV risks by treatment of hypertension, glycemic control, and lowering of serum cholesterol. In our previous in vitro and in vivo studies, administration of black raspberry extracts lowered blood pressure in rats and improved lipid profiles and vascular endothelial function in patients with metabolic syndrome. Some other studies have shown that black raspberry improves endothelial cell function and the process of atherosclerosis.^7–9 Anti-inflammatory and antioxidant properties of black raspberry could improve endothelial function and eventually increase circulating EPCs and decrease arterial stiffness. We hypothesized that administering black raspberry extract could have favorable effects on blood pressure, vascular endothelial function, and circulating EPCs. Therefore, we performed a prospective randomized study investigating the short-term effects of black raspberry on the circulating numbers of EPCs, central blood pressure, augmentation index, and inflammatory markers in patients with metabolic syndrome.

Materials and Methods

Study patients Patients were eligible for this study if they were between 18 and 75 years old with metabolic syndrome. For patients to meet the diagnostic criteria for metabolic syndrome, ≥3 of the following measurements had to be fulfilled: abdominal circumference ≥90 cm in men or ≥85 cm in women, triglyceride level ≥150 mg/dL, high-density lipoprotein cholesterol <40 mg/dL in men or <50 mg/dL in women, systolic blood pressure (SBP) of ≥130 mmHg, diastolic blood pressure (DBP) ≥85 mmHg, and fasting blood glucose of ≥100 mg/dL.^23,24 Participants underwent randomization at a 1:1 ratio to receive placebo or black raspberry extract for 12 weeks (Supplementary Table S1).
A total of 116 patients with metabolic syndrome were screened for inclusion in the study at Korea University Anam Hospital Cardiovascular Center from August 2013 to November 2013 (Fig. 1). Exclusion criteria were (i) patients who did not fulfill the inclusion criteria (n = 5), (ii) who did not provide informed consent (n  = 48), (iii) familial hypercholesterolemia, (iv) hepatic dysfunction (aspartate aminotransferase or alanine aminotransferase>twice the upper limit), (v) gastrointestinal disorder such as Crohn's disease or history of surgery, (vi) alcohol abuse, (vii) steroid or hormone replacement therapy, (viii) serum creatinine >2.0 mg/dL, and (ix) expected life expectancy of <1 year. Patients who had CV or cerebrovascular disease, such as coronary artery disease, heart failure, and stroke, were eligible for this study. Eligible patients (n = 51) were prospectively randomized into the black raspberry group (n = 26, 750 mg/day equivalent of 4 capsules/day) or placebo group (n  = 25) during the 12-week follow-up. Dried unripe black raspberries were made into capsules containing black raspberry powder under good manufacturing practices, and each black raspberry capsule contained 187.5 mg of dried unripe black raspberry powder. The participants were told to take a total of four black raspberry capsules each day or placebo during the 12-week follow-up.

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FIG. 1.  Study patients.

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Complete clinical workup was scheduled at baseline and at a 12-week follow-up. Central blood pressure, radial artery augmentation index, and the circulating number of EPCs were compared between the two groups during the follow-up. The primary endpoints of the study were to compare the short-term effects of black raspberry on the radial artery augmentation index and the circulating number of EPCs in patients with metabolic syndrome during the 12-week follow-up. Patients received randomization numbers sequentially from a secret randomization list that was computer generated in blocks of three by individuals who had no contact with the persons who assigned patients to study groups or performed any assessments on patients. The clinical research center was given a single-sealed opaque envelope for each patient that contained the treatment code and it was to be opened only in a medical emergency. Investigators and participants were unaware of the randomization assignments until the final data were obtained. All participants were instructed to follow a diet based on “Dietary Approaches to Stop Hypertension” (DASH) diet for controlling metabolic syndrome and were instructed not to consume any berry species. Telephone interviews were conducted to check adherence to the intervention by counting the remaining black raspberry pills biweekly. Remaining black raspberry capsules were counted during the follow-up, and patients who took more than 85% of the black raspberry capsules were considered compliant. The study was approved by the University Hospital Institute Review Board, and written informed consent was obtained from all participants or their legal guardians.

Preparation of unripe R. occidentalis extract

The unripe fruits of R. occidentalis were collected from the Gochang (Jeollabuk-Do) area in South Korea. In brief, fruits were extracted twice with tap water at 100°C using a reflux condenser. Black raspberry was extracted with a reflux condenser device by adding 10-fold solvent volume of the unripe black raspberries (water, 25%, 50%, and 75% ethanol) for 2 h. The extracts were filtered and concentrated, and the concentrates were lyophilized in a freeze dryer (PVTFD10R; Ilshinbiobase, Dongducheon, Korea). Total polyphenol content was measured by the Folin–Denis method, and the total flavonoid content was measured by the Davis method. Ellagic acid was used as a marker compound to develop suitable identification test for raw materials.²⁵ Ellagic acid (analytical standard, purity 95%; Sigma Co. St. Louis, MO, USA) was diluted with methanol. Ellagic acid was assayed by high-performance liquid chromatography (ACQUITY H-class; Waters, Co., Milford, MA, USA). One capsule of the black raspberry extract consisted of black raspberry powder (62.5%), magnesium stearate (1.5%), silica (1.5%), and isomaltose (34.5%). Placebo capsules had the same appearance, but contained isomaltose (97.0%), magnesium stearate (1.5%), and silica (1.5%). Isomaltose was made from corn powder. The nutrient composition in 100 g black raspberry was 9.6 g carbohydrate, 5.3 g fiber, 4.9 g sugar, 1.4 g protein, 0.5 g lipid, 29 mg calcium, 0.62 mg iron, 20 mg magnesium, 22 mg phosphorus, 21 mg vitamin C, 0.02 mg thiamin, 0.03 mg riboflavin, 0.6 mg niacin, 25 μg folate, 11 μg vitamin A, 128 μg β-carotene, 214 IU vitamin A, 1.17 mg vitamin E, 1.34 mg γ-tocopherol, 100 mg cyanidin, 0.4 mg pelargonidin, 37.1 mg catechin, 4.7 mg epicatechin, 3.6 mg quercetin, 0.7 mg myricetin, and 19.5 mg proanthocyanidins (USDA National Nutrient Database 2015). Further details of manufacture and characteristics of black raspberry powder were discussed in a previous study.¹¹

Measurements of circulating EPCs

Peripheral blood samples (4 mL) were drawn into heparinized tubes in the morning after an overnight fast. Peripheral blood mononuclear cells were isolated within 1 h by density gradient centrifugation using Ficoll-Paque Plus (17-1440-03; Amersham Biosciences, Piscataway, NJ, USA) and stored at 4°C until the cells were analyzed. For flow cytometry analysis, the cells were washed with phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) and were double stained with anti-CD34-FITC (348053; BD Pharmingen, San Diego, CA, USA) and anti-KDR-PE (FAB357P; R&D Systems, Minneapolis, MN, USA) or anti-CD34-FITC and anti-CD117-PE (555714; BD Pharmingen) or anti-CD34-FITC and anti-CD133-PE (130-080-801; Miltenyi Biotec, Bergisch Gladbach, Germany) monoclonal antibodies diluted 1:100 in PBS containing 2% FBS for 20 min at 4°C. A negative control was also stained with FITC mouse IgG1 isotype control (555909; BD Pharmingen) and PE mouse IgG1 isotype control (349043; BD Pharmingen) antibodies. After washing with PBS containing 2% FBS, the cells were resuspended and analyzed by flow cytometry. For double-staining experiments, the interference of two fluorescence channels was adjusted by compensation. Three thousand cells per sample were analyzed on a FACS Vantage SE flow sorter (BD Biosciences, San Jose, CA, USA). Dead cells and debris were gated out using scatter properties of the cells. Data were analyzed by using CellQuest Pro software (BD Biosciences). CD34⁺KDR⁺ or CD34⁺CD117⁺ or CD34⁺CD133⁺ double-positive cells were defined as circulating EPCs after gating on lymphocyte population. The number of positive cells was calculated on the basis of absolute leukocyte count × percentage (%) of positive cells and expressed as the absolute number of cells per 1 mL of whole blood (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/jmf).

Measurements of central blood pressure and augmentation index

Central pressure recordings were obtained using an Omron HEM-9000AI (cSBP-Omron) as described by the manufacturer's user manual. Blood pressure measurement was obtained through the digital oscillometric method using a blood pressure cuff. Accompanying augmentation index calculations were made based on the patient‘s pressure waveforms calibrated using brachial SBP and DBP. The augmentation index is determined by the change in pressure between the first and second peaks divided by the pulse pressure (augmentation index = ΔP/PP). The first peak is obtained when blood is ejected from the aorta. The second pressure peak occurs when blood is reflected at the aortic bifurcation. The pulse pressure is the overall peak pressure. All data were stored and analyzed offline after completion of testing.

Laboratory analysis

Inflammatory markers such as high-sensitive C-reactive protein (hsCRP), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were measured for both groups at the beginning of the study and at the 12-week follow-up visit. Venous blood samples were drawn from each patient after 8 h or overnight fasting. Blood samples were centrifuged to obtain plasma, which was stored at −80°C. TNF-α was measured by a sandwich enzyme-linked immunosorbent assay (ELISA) with a minimum detectable level of 0.5 pg/mL (ALPCO Diagnostics, Salem, NH, USA). Undetectable TNF-α values were recorded as 0.4 pg/mL. High-sensitivity IL-6 was measured by a sandwich ELISA with a minimum detectable level of 0.16 pg/mL (ALPCO Diagnostics). hsCRP concentration was quantified using the latex nephelometer II (Dade Behring, Inc., Newark, DE, USA). The plasma adiponectin concentration was assessed by the radioimmunoassay (Linco Research, Inc., St. Charles, MO, USA). The sensitivity of this assay was 0.78 ng/mL. The coefficient of variation for intra- and interassay was 9.3% and 15.3%, respectively. In addition, sICAM-1 and sVCAM-1 were measured using ELISA according to the manufacturer's instructions (R&D Systems).

Statistical analyses

Data are expressed as means for continuous variables, and data for the categorical variables are expressed as the number and the percentage of patients. Chi-square tests were used for categorical variables. The change from baseline was calculated as the value obtained at the end of the treatment subtracted from the value obtained at the beginning of the study. The results between two groups were compared by an unpaired Student's t-test, and the comparisons between before and after treatment were analyzed by a paired t-test. Using a two-sided test for differences in independent binomial proportions with an α level of 0.05, we calculated that 46 patients (23 patients for each group) would have to undergo randomization for the study to have 80% power to detect a difference in the circulating number of EPCs between the two groups; therefore, we enrolled 51 patients to account for 10% loss in the 12-week follow-up. Variables that did not show normal distribution were log transformed in subsequent analysis. P-value <.05 was considered statistically significant. SPSS software (version 20.0) was used for the analyses.

Results

Study patients characteristics Of the 116 patients that underwent actual screening, the eligible patients (n = 51) were prospectively randomized into the black raspberry group (n = 26, 750 mg/day equivalent of 4 capsules/day) and placebo group (n  = 25) and were followed for the 12-week study period at Korea University Anam Hospital Cardiovascular Center from August 2013 to November 2013. Baseline patient characteristics such as mean ages and body mass indexes were similar between the two groups (Table 1). Risk factors such as hypertension, diabetes, hyperlipidemia, and current smoking were not significantly different between the two groups. There were no patients who had coronary artery diseases or cerebrovascular diseases in this study. Moreover, medications at baseline did not differ significantly between the two groups. During the intervention, none of the patients dropped out. No adverse event was reported.

Table 1. Baseline Patient Characteristics

Changes in central blood pressure and augmentation index There were no significant changes in SBP, DBP, or central systolic blood pressure. However, the radial artery augmentation index decreased significantly in the black raspberry group when compared to the placebo group (−5% ± 10% vs. 3% ± 14%, P < .05) (Table 2). The radial artery augmentation index decreased significantly even after adjusting for age (P = .005). In subgroup analysis, sex did not significantly impact the augmentation index (P = .066).

Table 2. Changes in Central Blood Pressure and Radial Augmentation Index

Circulating EPCs and inflammatory parameters during the 12-week follow-up Counts of circulating EPCs at baseline were similar between the two groups in CD34/KDR⁺, CD34/CD117⁺, and CD34/CD133⁺ cells. However, increases in CD34/CD133⁺ cells at 12-week follow-up were significantly greater in the black raspberry group when compared to the placebo group (19 ± 109/μL vs. −28 ± 57/μL, P < .05) (Table 3). Decreases from the baseline in IL-6 and TNF-α were significantly greater in the black raspberry group than in the placebo group (−0.5 ± 1.4 pg/mL vs. −0.1 ± 1.1 pg/mL, P < .05, and −5.4 ± 4.5 pg/mL vs. −0.8 ± 4.0 pg/mL, P < .05, respectively). Increases from the baseline in adiponectin levels (2.9 ± 2.1 μg/mL vs. −0.2 ± 2.5 μg/mL, P < .05) were significantly greater in the black raspberry group.

Table 3. Changes in Circulating Endothelial Progenitor Cells and Inflammatory Parameters During 12-Week Follow-Up

Discussion

This is the first prospective randomized double-blind study to investigate the effect of black raspberry on circulating EPCs and on arterial stiffness. Administering black raspberry (R. occidentalis) extract, 750 mg/day for 12 weeks, significantly lowered the radial artery augmentation index compared to the placebo group and significantly increased the circulating numbers of CD34/CD133⁺ cells during the follow-up.
The augmentation index is defined as the percentage of central pulse pressure attributed to reflected wave overlap in systole and is related with CV outcomes.^26,27 When arteries are stiff, the reflected wave overlaps earlier with incident wave, resulting in increased pulse pressure and higher augmentation index.²⁸ Arterial stiffness is a result of endothelial damage since endothelial dysfunction causes decreased production and function of NO and reduces arterial compliance.²⁹ Increased oxidative stress and inflammation also exacerbate arteriolar remodeling.^30,31 Although central blood pressure did not change in this study, the decreases in augmentation index were observed only in the black raspberry group, which could be explained by the improvement in vascular endothelial function in the black raspberry group from our previous study.¹⁰ The endothelium regulates vascular reactivity through the dilator-associated mediators including NO and prostaglandins.^32,33 Flavonoids, major components of the black raspberry, are a well-known element that improves vascular function through the endothelium-dependent flow-mediated vasodilation and reduces CV risks in patients with endothelial dysfunction.^10,34 Flavonoids increase bioavailability of NO and endothelial nitric oxide synthase (eNOS).^35,36 Moreover, an experimental study shows that polyphenolic compounds can induce the endothelium-NO-dependent relaxation of coronary arteries.³⁷ In particular, anthocyanin, one of the phenolic compounds, possesses anti-inflammatory and antioxidant capacity and improves vascular function by upregulating NO and eNOS.^38–40 A study has shown that administration of anthocyanins improves arterial stiffness such as the augmentation index and pulse wave velocity, arterial systolic pressure, and central blood pressure,^41,42 leading to the assumption that anthocyanins reduce the risks of CV disease-related mortality.⁴³ The improvement of augmentation index in this study might have been mediated by the vasodilating and anti-inflammatory effects of black raspberry.
Decrease of the augmentation index is presumably associated with the circulating number of EPCs. EPCs are bone marrow-derived stem cells that are mobilized into peripheral circulation. These cells stimulate neoangiogenesis and repair the damaged vascular endothelium.⁴⁴ EPCs home into the injury site to replicate endothelial cells and activate the endogenous repair system.⁴⁵ EPCs represented with circulating CD34⁺ and CD133⁺ cells express vascular endothelial growth factor receptor 2 (VEGFR-2) and eNOS.⁴⁶ CD34⁺ cells also stimulate subject angiogenic cytokines.⁴⁷ Some studies have demonstrated that EPCs restore ischemic damage in vivo and improve clinical outcomes.^48,49 The number of circulating EPCs inversely correlates with CV risk factors, suggesting that the circulating number of EPCs is lower in patients with CV diseases.^21,50 Moreover, patients with increased numbers of circulating EPCs have preserved endothelial function and show better arterial stiffness profiles regardless of the risk factors.²¹ Increases in circulating EPCs during the 12-week follow-up in this study suggest rapid restoration to the damaged endothelium, thereby contributing to the improvement of arterial stiffness and the augmentation index. Other randomized clinical trials of cranberry juice and blueberry drink did not show significant changes in the augmentation index.^51,52 However, administering black raspberry contributed to the increases in the numbers of circulating EPCs, which presumably improved endothelial dysfunction and upregulated eNOS and NO expression, thereby improving arterial stiffness and the augmentation index in patients with metabolic syndrome in this study.
Our study had a few limitations. The total number of study participants was relatively small for evaluating clinical CV events. Black raspberry contains various types of beneficial natural compounds, including polyphenolic compounds; however, exact mechanisms about the relationship between the beneficial compounds in black raspberry and favorable effects on circulating number of EPCs and arterial stiffness need to be further investigated.

In conclusion, the use of black raspberry significantly lowered the augmentation index and increased circulating EPCs, thereby improving CV risks in patients with metabolic syndrome during the 12-week intervention.