*3.1. Antihypertensive E*ff*ects*

Hypertension increases the risk of cardiovascular disease [84]. Antihypertensive compounds in foods have been identified using a stroke-prone spontaneously hypertensive (SHRSP) rat model, which is genetically predisposed to hypertension and cerebral stroke [85]. Sansawa et al. [86] investigated the e ffects of dried *Chlorella* powder (*C. regularis*) on blood pressure, cerebral stroke lesions, and the life span of SHRSP rats. In 12-week-old SHRSP rats fed *Chlorella* (5%, 10%, and 20%) for 13 weeks, elevated blood pressure significantly decreased in the 10% and 20% *Chlorella* groups compared with the untreated controls. Serum total cholesterol levels were significantly lower in all *Chlorella* groups, and their average life span was more than that of the controls. To characterize the antihypertensive compounds in *Chlorella*, *Chlorella* powder was fractionated into hot-water-soluble, lipid-soluble, and residual fractions. Blood pressure was significantly lower in rats fed the lipid or residual fraction but not in those fed the hot-water-soluble fraction. The lipid fraction contained substantial amounts of carotenoids, which are potent antioxidants, and phospholipids, which mediate aorta collagen and elastin metabolism. The residual fraction contained a high level of arginine, which increases the production of endothelium-derived relaxing factor. These beneficial e ffects of *Chlorella* powder on SHRSP rats might result from synergism between its numerous bioactive compounds.

To evaluate whether daily *Chlorella* supplementation can reduce blood pressure in subjects with mild to moderate hypertension, a pilot study was conducted in 24 participants administered *C. pyrenoidosa* (10 g of *Chlorella* tablets and 100 mL *Chlorella* extract) [87]. After two months of *Chlorella* supplementation, the average heart rate and sitting systolic and diastolic blood pressures only slightly changed. On the other hand, for some subjects with mild to moderate hypertension, *Chlorella* supplementation reduced or maintained their sitting diastolic blood pressure.

Arterial sti ffness is a well-established risk factor of cardiovascular disease [88]. Previous studies have reported that antioxidants [89], potassium [90], and *n*-3 unsaturated fatty acids [91] decrease arterial sti ffness. Nitric oxide (NO), derived from arginine in the vascular endothelium, is an important modulator of arterial sti ffness [92]. *Chlorella* products contain antioxidants, vitamins, potassium, arginine, and *n*-3 unsaturated fatty acids. To evaluate the e ffects of *Chlorella* supplementation on arterial sti ffness, a single-blinded, placebo-controlled crossover study was conducted in 14 young participants who were administered *C. pyrenoidosa* (6 g/day) or placebo for four weeks, with a 12-week washout period between trials, in a randomized order [93]. No di fferences were observed in blood pressure or heart rate before and after supplementation in both the placebo and *Chlorella* groups. Brachial-ankle pulse wave velocity, a measure of arterial sti ffness, decreased in the *Chlorella* group but not in the placebo group [93]. A similar trial in 32 middle-aged and older subjects reports that the brachial-ankle pulse wave velocity decreased after *Chlorella* supplementation but not after placebo supplementation [94]. These changes in brachial-ankle pulse wave velocity with *Chlorella* supplementation correlated with the plasma NOx level. These results sugges<sup>t</sup> that *Chlorella* supplementation decreases arterial sti ffness in both younger and older subjects.

The e fficacy of *Chlorella* supplementation in reducing cardiovascular risk factors was assessed in a meta-analysis of 19 randomized controlled trials including 797 subjects [95]. This study concluded that *Chlorella* supplementation improves total cholesterol levels, low-density lipoprotein cholesterol levels, systolic blood pressure, diastolic blood pressure, and fasting blood glucose levels but not triglyceride levels, high-density lipoprotein cholesterol levels, and body mass index.

#### *3.2. Antihypercholesterolemic and Antihyperlipemic E*ff*ects*

Elevated total cholesterol and triglycerides and abnormal metabolism of lipoproteins and apolipoproteins are responsible for an increased risk of cardiovascular disease [96–98]. The indigestible components of foods, such as dietary fiber, decrease serum cholesterol by inhibiting the intestinal absorption of neutral steroids [99]. *Chlorella* supplementation reportedly decreases serum cholesterol levels in model animals [100]. To identify the bioactive compounds responsible for this e ffect, the indigestible fraction of *C. regularis* powder was isolated and characterized, revealing a content of 43% crude protein, 37.3% dietary fiber, 6.9% carbohydrate, 5.4% moisture, 4.3% crude fat, and 2.7% ash [101]. Rats fed a diet with 5.3% of this indigestible fraction demonstrated lower serum and liver cholesterol levels and higher fecal neutral steroid levels as compared with those fed a diet of 12.7% *Chlorella* powder. Both *Chlorella* powder and the indigestible fraction exhibited a high bile-acid binding capacity in vitro. Furthermore, the indigestible fraction increased the hepatic mRNA levels of cholesterol <sup>7</sup>α-hydroxylase, which is the rate limiting enzyme for cholesterol catabolism and bile-acid synthesis [102]. These results indicate that the indigestible fraction of *Chlorella* possesses hypercholesteromic activity, which improves cholesterol catabolism via the upregulation of hepatic cholesterol <sup>7</sup>α-hydroxylase expression.

*Chlorella* supplementation is also reported to decrease serum cholesterol levels in hyperlipemia and mild hypercholesterolemic patients in a small, open-label trial [103]. To evaluate the preventive role of *Chlorella* in maintaining serum cholesterol levels against excess dietary cholesterol intake, a double-blind, randomized, placebo-control study was conducted in 63 mildly hypercholesterolemic subjects treated with either *C. vulgaris* (5 g/day) or placebo for four weeks [104]. A similar trial investigated cholesterol levels in 34 participants administered 510 mg of dietary cholesterol from three eggs concomitantly with either *Chlorella* (*C. vulgaris*) (5 g/day) or a matched placebo for 4 weeks [105]. Participants on the three-egg diet alone exhibited significant elevation in serum total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol levels. The administration of *Chlorella* in addition to the three-egg diet significantly suppressed these increases in total cholesterol and low-density lipoprotein cholesterol levels and significantly increased serum lutein and α-carotene levels [105]. In mildly hypercholesterolemic subjects, *Chlorella* administration resulted in marked changes in total cholesterol, triglycerides, lutein/zeaxanthin, and α-carotene levels as well as a significant decrease in very low-density lipoprotein cholesterol, apolipoprotein B, non-high-density lipoprotein, and high-density lipoprotein/triglyceride levels [104]. These results sugges<sup>t</sup> that *Chlorella* might inhibit the intestinal absorption of dietary and endogenous lipids. In addition, the observed changes in serum lipids may be associated with changes in serum carotenoids. These results sugges<sup>t</sup> that daily consumption of *Chlorella* provides potential health benefits by reducing the levels of serum lipid risk factors, such as triglycerides and total cholesterol, in mild hypercholesterolemic subjects.
