Multicomponent supplement containing Chlorella decreases arterial stiffness in healthy young men.

Chlorella, a unicellular green alga, contains various antioxidants and other nutrients such as amino acids and fiber. Previous studies have reported that supplementation with multiple antioxidants reduces arterial stiffness, a well-established cardiovascular risk factor. We investigated the effects of Chlorella intake on arterial stiffness using a single-blinded, placebo-controlled crossover study design. Fourteen young men took placebo or Chlorella tablets for four weeks, with a 12-week washout period between trials, in a randomized order. Before and after each trial, blood pressure, heart rate, and brachial-ankle pulse wave velocity, an index of arterial stiffness, were measured. Treatment compliance was comparable between the two groups. There were no differences in blood pressure and heart rate before and after supplementation in both the placebo and Chlorella groups. Brachial-ankle pulse wave velocity decreased after Chlorella intake (before vs after intake; 11.6 ± 0.2 vs 11.1 ± 0.1 m/s, p = 0.01), but not after placebo intake (11.4 ± 0.2 vs 11.4 ± 0.2 m/s, p = 0.98). Multicomponent analysis of the Chlorella-containing tablet detected nutrients that can reduce arterial stiffness, such as antioxidant vitamins, arginine, potassium, calcium, and n-3 unsaturated fatty acids. These results suggest that intake of a Chlorella-containing multicomponent supplement can decrease arterial stiffness.

RCT Entities:   Population Interventions Outcomes

Introduction

Arterial stiffness is a well-established risk factor for cardiovascular disease.( Previous studies have reported that some nutrients such as antioxidants,( isoflavone,( n-3 unsaturated fatty acids,( lactotripeptides,( and potassium( decrease arterial stiffness. However, interindividual differences in nutritional status may result in differences in the effects of various nutrients on arterial stiffness. Additionally, beneficial effects are more definitively seen when nutrients are taken in combination than in isolation. For example, combination treatment in men (1 g vitamin C and 400 IU vitamin E per day for 8 weeks) and the addition of antioxidants in women (120 mg vitamin C, 30 mg vitamin E, 6 mg beta-carotene, 100 µg selenium, and 20 mg zinc per day for 7.2 years) decreased aortic pulse wave velocity (PWV),( an index of arterial stiffness, whereas supplementation with vitamin C alone in men (500 mg daily for 30 days) and vitamin E alone in women (400 IU daily for 10 weeks) did not decrease aortic PWV.( In this context, a multicomponent supplement suitable for individuals with different nutritional status is of significance.

The use of natural ingredients in a multicomponent supplement may be favorable, although it is not easy to determine the best nutritional composition of a dietary supplement. Chlorella is a unicellular green alga that grows in fresh water. Since it contains various antioxidants and other nutrients, Chlorella-containing multicomponent supplementation may help reduce arterial stiffness. However, the effect of Chlorella-containing supplements on arterial stiffness remains to be elucidated. We conducted a single-blinded crossover study to investigate effects of placebo and Chlorella supplementation on arterial stiffness. It is difficult to discriminate between reduced arterial stiffness and decreased blood pressure when both measures decrease simultaneously. Additionally, the menstrual cycle influences arterials stiffness.( Hence, we recruited normotensive young men to investigate the effect of Chlorella intake on arterial stiffness without blood pressure and menstrual cycle changes as confounding factors.

Materials and Methods

Participants

Fourteen men volunteered to participate in this study; none of them use dietary supplements on a regular basis. There were four subjects who engaged in recreational exercise regularly (more than 30 min/day and 3 days/week) and two subjects who were kendo and futsal athletes (vigorous exercise 90–140 min/day and 5–6 days/week), respectively. The participants were asked not to modify their regular lifestyle while taking the tablets during the trial periods. None of the participants had any symptoms or history of overt chronic disease. None of the subjects were smokers or were taking any medication. The mean ± SE age and height values were 20.2 ± 0.3 years and 1.67 ± 0.01 m, respectively.

The present study was approved by the Ethical Committee of the Institute of Health and Sport Sciences of the University of Tsukuba. This study conformed to the principles outlined in the Helsinki Declaration. All participants gave their written informed consent before their participation in this study.

Experimental design

Each participant took part in two supplement trials, placebo and Chlorella, in a randomized order. First, blood pressure and brachial-ankle PWV (baPWV), an index of arterial stiffness which has been widely used in Japan and other countries over the past 10 years,( were measured as in our previous study.( The following day, the participants were randomized into either the placebo or Chlorella (SunChlorella A; Sun Chlorella, Kyoto, Japan) group in a single-blinded manner, to be taken at a dose of 30 tablets per day (15 tablets twice daily, after breakfast and dinner) for four weeks.( This is in accordance with the general recommended dosage for Japanese consumers. Second, blood pressure and baPWV measurements were taken one day after the final tablet intake. Compliance with the prescription was documented via intake logs. After a washout period of 12 weeks, the second trial commenced with the alternate group assignment.

Placebo and Chlorella tablets

The placebo and Chlorella tablets used in this study were the same as in our previous studies.( The mass of each tablet was 243 mg and 200 mg, respectively. The main components of the placebo tablet were lactose and colorant. The main ingredient of Chlorella tablet was dried Chlorella pyrenoidosa powder. The respective nutritional values per 100 g of the placebo and Chlorella tablets were: energy, 406 and 399 kcal; water, 3.2 and 5.3 g; protein, 2.0 and 60.8 g; lipids, 5.9 and 9.2 g; sugar, 85.6 and 6.3 g; dietary fiber, 1.1 and 11.9 g; and ash, 2.2 and 6.5 g. The color and shape of the placebo and Chlorella tablets were similar. Additional multicomponent analysis of the Chlorella-containing tablet was entrusted to the Japan Dairy Technical Association (Tokyo, Japan); the analyses were performed using high performance liquid chromatography, atomic absorption spectrophotometry, and other methods.

BaPWV and blood pressure

All subjects were asked to refrain from intense physical activity or exercise for a 24 h period prior to measurements, which were made after an overnight fast. After a resting period in a quiet, temperature-controlled room, baPWV was measured as in our previous studies.( Briefly, brachial and post-tibial artery pressure waveforms were obtained simultaneously, in triplicate, by cuffs connected to an air-plethysmographic sensor (form PWV/ABI; Omron Colin, Tokyo, Japan). The pulse-wave travel distance was calculated from the subject’s height and the travel time was automatically determined from the waveforms. BaPWV was calculated as the travel distance divided by the travel time. In our laboratory, the representative day-to-day coefficient of variation for baPWV was 2.2 ± 1.3%.( Blood pressure was measured using oscillometry and heart rate (HR) was calculated from an electrocardiogram (form PWV/ABI, Omron Colin).

Statistical analysis

Results are given as means ± SE. P values of <0.05 were considered statistically significant. We tested whether the variables were normally distributed using the Kolmogorov-Smirnov test. Differences in patient compliance and change in baPWV between placebo and Chlorella trials were tested using t tests. To compare the effects of Chlorella intake with placebo supplementation, a repeated-measures two-way (placebo or Chlorella × intake period) analysis of variance (ANOVA) was used. When a significant interaction was observed, one-way ANOVA and a post-hoc Bonferroni-Dunn test were used to identify the effects of placebo and Chlorella intake.

Results

All of the variables before and after intake were normally distributed in both groups. Compliance with the group assignment was comparable between the placebo and Chlorella groups (97.3 ± 1.0 vs 96.7 ± 1.2%; t = 0.5, p = 0.65). There were no trial × intake period interactions and no effects of group and intake were observed with respect to body weight (before vs after intake: placebo group, 65.6 ± 2.2 vs 66.3 ± 2.2 kg and Chlorella group, 65.3 ± 2.3 vs 65.7 ± 2.4 kg; F = 0.4 and p = 0.50), blood pressure, and HR (Table 1). There was a significant interaction in baPWV between group and intake period (F = 4.5, p = 0.04; Fig. 1A); baPWV decreased from baseline after Chlorella intake (F = 7.1, p = 0.01) while placebo tablets had no effect (F = 0.0, p = 0.98). The change in baPWV with a four-week trial was greater in the Chlorella group than in the placebo group (Fig. 1B). In the multicomponent analysis of the Chlorella-containing tablets, numerous nutrients were detected, some of which have been previously shown to lower arterial stiffness (Table 2).

Table 1

Blood pressure and heart rate before and after Chlorella intake

		Before	After	Interaction
Systolic blood pressure (mmHg)	Placebo	117 ± 2	118 ± 2	F = 0.4
	Chlorella	119 ± 2	119 ± 2	p = 0.54
Diastolic blood pressure (mmHg)	Placebo	68 ± 2	68 ± 2	F = 0.0
	Chlorella	68 ± 1	67 ± 1	p = 0.90
Heart rate (bpm)	Placebo	59 ± 2	59 ± 2	F = 0.1
	Chlorella	59 ± 2	59 ± 2	p = 0.77

Values are means ± SE.

Fig. 1

Arterial stiffness before and after Chlorella intake. Values are means ± SE. baPWV: brachial-ankle pulse wave velocity.

Table 2

Primarily results of multicomponent analysis of Chlorella tablet

Total		Detail
Nutrient	Value	Nutrient	Value
Protein (g/100 g)	60.8	Arginine (g/100 g)	3.16
Carbohydrate (g/100 g)	18.2	Dietary fiber (g/100 g)	11.9
Lipid (g/100 g)	9.2	n-3 unsaturated fatty acid (%)	8.6
Ash (g/100 g)	6.5	Potassium (mg/100 g)	970
		Magnesium (mg/100 g)	370
		Calcium (mg/100 g)	330
		Beta-carotene (mg/100 g)	4.3
		Vitamin C (mg/100 g)	4.0
		Vitamin E (mg/100 g)	2.7
		Zinc (mg/100 g)	1.1

In the multicomponent analysis of the Chlorella-containing tablets, numerous nutrients were detected, some of which have been previously shown to lower arterial stiffness. A portion of this table is republished from our previous study.(

Discussion

This study investigated the effect of four weeks of a Chlorella-derived multicomponent supplement on baPWV using a single-blinded, randomized, crossover study design. The salient finding is that baPWV decreased from baseline after Chlorella supplementation, whereas no changes occurred when subjects took placebo. Additionally, many nutrients that can decrease arterial stiffness were detected in the multicomponent analysis of the Chlorella tablet. These results suggest that oral supplementation with a multicomponent supplement containing Chlorella decreases arterial stiffness.

Arterial stiffness and blood pressure affect each other; decreased blood pressure reduces arterial stiffness and vice versa. To investigate effects of lifestyle modification such as exercise on arterial stiffness in a blood pressure-independent manner, previous studies recruited normotensive individuals and demonstrated a decrease in arterial stiffness without blood pressure reduction.( Similarly, there were no changes in blood pressure in both groups since we recruited normotensive individuals to rule out the effects of blood pressure on arterial stiffness. Additionally, women were not recruited to exclude the effects of the menstrual cycle on arterial stiffness.( There were no smokers or subjects on medication in this study. These relative strengths of this study help isolate the effects of the Chlorella-containing tablets on arterial stiffness. Given the Bogalusa Heart Study’s finding that carotid artery intima-media thickness is associated with the cumulative burden of cardiovascular risk factors starting in childhood,( distensible central arteries would be preferable not only in older individuals but also younger individuals. However, it is unclear whether the present finding is applicable to older individuals and women. Further studies are warranted as next steps.

Arterial stiffness is determined by structural (e.g., elastin and collagen content in the arterial media) and functional (e.g., endothelial function and sympathetic nervous system activity) factors. However, more time is needed to elicit structural changes than functional changes. It is reasonable to assume that reductions in arterial stiffness during a four-week period were due more to functional changes. For instance, potassium can affect arterial stiffness by decreasing the contractility of vascular smooth muscle cells via sodium excretion.( Arterial stiffness can be reduced by combined intake of antioxidant vitamins (i.e., vitamin C and E) through improvements in vascular endothelial function.( n-3 Unsaturated fatty acids can improve arterial stiffness( by the antiinflammatory function.( In addition, arginine can be converted to nitric oxide, an endothelium-derived vasodilatory factor, which decreases arterial stiffness.( However, it is unclear which mechanisms were responsible for the observed reduction in arterial stiffness in this study. A comprehensive investigation is necessary to determine the mechanisms responsible for the beneficial effects of Chlorella supplementation.

This study has several limitations. First, the subjects were normotensive young men as mentioned above. Additionally, the sample size was not large. Larger studies in older and hypertensive individuals are the next steps. Second, we could not investigate the mechanisms underlying the Chlorella-induced declines in arterial stiffness although we performed multicomponent analysis and discussed some possibilities. Third, this study could not control for the dietary habits of each subject that could influence the effects of Chlorella intake. For example, the effects may be less marked in individuals who are following diet restriction than counterparts because phosphate restriction improves endothelium function and sodium restriction reduces arterial stiffness.( Finally, even though the dosage was in accordance with the current recommendations for consumers, the number of tablets taken per day was very high. A study to determine the optimal dosage is needed.

In conclusion, the present results suggest that intake of a Chlorella-containing multicomponent supplement decreases arterial stiffness in young normotensive men.