Table 1.

Characteristics of animal studies related to hyperlipidemia included in the review.

Study	Disease/ Condition	Study Type / SQ Source	Sample/ Population	Methodology	Results (SQ and CVD Only)	Comments/Outcomes
Study 1 Khor and Chieng 1997 [48]	Hyperlipidemia	Animal study   Obtained from Sigma, St. Louis, MO, USA	Male Syrian hamsters (123 ± 2 g)   The animals were randomly assigned into four groups.	The hamsters were fed with semi-synthetic diet containing 20% (w/w) of fat and a palm oil triacylglycerol (POTG) fraction isolated from commercial palm olein. They were divided into 4 groups: a) POTG b) POTG + SQ (POTG-SQ) c)POTG + SQ + pure tocotrienols (POTG-SQ-T3) d) POTG+ SQ and α-tocopherol (POTG-SQ- αT).   The animals were fed accordingly for 45 days. SQ, T3, and α-T were supplemented at concentrations of 0.1%, 162 ppm, and 72 ppm, respectively.   At the end of the treatment period, fasting blood samples were taken for the measurement of serum TC, HDL-C, TAG, and LDL-C. The livers were excised for measurement of liver lipids.	Serum: SQ-alone supplementation had significantly lowered (p < 0.05) serum TC levels as compared to the POTG group SQ-alone supplementation also lowers HDL-C, LDL-C, and TAG levels, but the reduction was not significant (p > 0.05) as compared to the POTG group.   Liver: SQ-alone supplementation had significantly increased liver total lipids (p < 0.0l) and TC levels (p < 0.05).   SQ-alone supplemented group had elevated the liver TAG level, while other liver lipids such as diacylglycerols (DAG), monoacylglycerols (MAG), free fatty acids (FFA), and phospholipids (PL) were not affected.	Squalene supplemented in short-term period (45 days) at a low level (0.1%) reduced serum TC.   The ability of squalene to lower serum TC might be enhanced by cholesterol esterification activity in the liver.
Study 2 Shin et al. 2004 [47]	Hyperlipidemia	Animal study Amaranth SQ (in-house extraction) Shark liver SQ (obtained from Sigma, St. Louis, MO)	Male Sprague Dawley rats (110–130 g) Randomly divided into three groups	All rats were fed with 1% cholesterol diet for four weeks to induce hypercholesterolemia and were assigned into i) group 1: injected with saline (control) ii) group 2: injected with amaranth SQ (AS) iii) group 3: injected with shark liver SQ (SS)   Both treatment groups, AS and SS, were injected at 200 mg/kg via intraperitoneal route for seven days prior to the sacrifice.   Fecal samples were collected for the last 3 days for analysis of steroid excretion. Blood samples were analyzed for serum and liver lipids. The liver was excised to obtain liver microsomes for HMG-CoA reductase activity.	AS injection to the rats caused significant(p < 0.05) decrease in serum (TC and TG) and hepatic (cholesterol and TG) profiles compared to control and SS groups. Serum HDL-C levels in the AS group were significantly increased, resulting in a 45% reduction in atherogenic index.   The AS group also significantly (p< 0.05) increased in fecal excretions of cholesterol and bile acid and slightly inhibited HMG-CoA reductase activity. However, these effects were not observed in SS-injected group.	Amaranth SQ (AS), which represents a plant source of SQ, exerts better hypolipidemic effects compared to the shark liver SQ (animal source of SQ). The authors concluded that the cholesterol-lowering effect of amaranth SQ might be mediated by the following: i)increased fecal elimination of steroids and ii)different sources of SQ (plant vs animal) that might affect cholesterol metabolism.
Study 3 Liu et al. 2009 [51]	Hyperlipidemia	Animal study   Obtained from Healthy Nature Resource Inc	40 male Wistar rats (22 days old)   Randomly divided into two groups (20 rats per group)	The two groups were fed with the following diet for four weeks: 1)control group received a diet without squalene and 2)SQ group was provided with high squalene diet (1000 mg/kg orally).   When the rats were 51 days old (day 0 after the SQ withdrawal) and 75 days old (day 24 after the SQ withdrawal), ten rats from each SQ and control group were sacrificed. Blood samples were collected for measurements of leptin, glucose, cholesterol, and triglycerides. Tail arterial blood pressure (BP) and body weight were monitored weekly.	Following SQ feeding, BP and body weight gain were lower in the SQ group.   BP was significantly lower from 47 days of age in the squalene-fed group compared to controls (p < 0.01).   Plasma leptin, glucose, cholesterol, and triglycerides in SQ fed rats group were significantly lower compared to the control group at 51 and 75 days of age (p < 0.05).	Squalene may counteract the increase in body fat, BP, and levels of plasma leptin, glucose, cholesterol, and triglycerides.   Squalene may act as an alternative treatment for clinical management of high blood pressure (BP) and obesity.
Study 4 Gabas-Rivera et al. 2014 [46]	Hyperlipidemia	Animal study   The source was not disclosed.	Male wild-type (WT), Apo E-deficient and Apoa1-deficient mice on C57BL/6J genetic background   Following baseline blood samples, groups with similar initial plasma cholesterol were established.	All mice were fed chow semi-purified diets and divided into 1) control groups: a) WT mice, n = 6; b) Apoa1-deficient mice, n = 7; and c) Apo E-deficient mice, n = 13 and 2) SQ-treated animals: a) WT mice received 1 g SQ/kg BW, n = 7; b) Apoa1-deficient mice received 1 g SQ/kg BW, n = 7; and c) Apo E-deficient animals were given two different doses of SQ: 0.25 g SQ/kg BW, n = 13 and 1 g SQ/kg BW, n = 14   The animals were fed the experimental diets for 11 weeks.   Following the intervention, blood was sampled for measurement: a) plasma parameters TC, TG, HDL-C, nonesterified fatty acids, and aryl esterase; b) lipoprotein profiles VLDL, LDL, and HDL; and c) oxidative stress variable ROS levels in lipoprotein fractions.	Plasma parameters WT mice: The supplementation of SQ at 1 g/kg BW significantly increased HDL-C, nonesterified fatty acids, and aryl esterase activity when compared to the control group (p < 0.05).   Apoa1-deficient mice: The supplementation of SQ at 1 g/kg BW significantly increased HDL-C and aryl esterase activity when compared to the control group (p < 0.05).   Apo E-deficient mice: The supplementation of SQ at 1 g/kg BW significantly increased HDL-C when compared to the control and 0.25 g/kg BW SQ-treated groups (p < 0.05).   The supplementation of SQ at 0.25 g/kg BW significantly increased total cholesterol when compared to the control and 1 g/kg SQ-treated groups (p < 0.01).   Lipoprotein profiles WT mice: The administration of SQ at 1 g/kg BW induced an increase in HDL cholesterol that was accompanied by increased HDL phosphatidylcholine, whilst no changes in sphingomyelin and Apoa1 and redistribution of Apoa4 towards smaller HDL particles were observed.   Apoa1-deficient mice: The administration of SQ at 1 g/kg BW did not have any effect on cholesterol distribution, decreased phosphatidylcholine, and increased Apoa4 in HDL particles and did not modify HDL sphingomyelin but decreased this phospholipid in LDL.   Apo E-deficient mice: The SQ administration at either studied dose had little effect on cholesterol, esterified cholesterol, phosphatidylcholine, and sphingomyelin. However, it increased the presence of Apoa4 in HDL and decreased LDL Apoa4.   Oxidative stress WT mice: The supplementation of SQ at 1 g/kg BW significantly reduced levels of ROS in isolated VLDL, LDL (p < 0.01), and HDL(p < 0.05) as well as plasma malondialdehyde (MDA) levels (p < 0.01) compared to the control group.   Apoa1-deficient mice: The supplementation of SQ at 1 g/kg BW significantly reduced levels of ROS in isolated LDL (p < 0.01) and HDL (p < 0.05) compared to the control group.   Apo E-deficient mice: The supplementation of SQ at 1 g/kg BW significantly reduced levels of ROS in isolated VLDL (p < 0.01) and HDL (p < 0.05) as well as plasma malondialdehyde (p < 0.01) compared to the control group.   The supplementation of SQ at 0.25 g/kg BW significantly reduced levels of ROS in isolated VLDL (p < 0.01), HDL (p < 0.01), and LDL + HDL (p < 0.05) as well as plasma malondialdehyde (p < 0.01) compared to the control group.	SQ supplemented at a high dose (1 g/kg) for a long-term administration (11 weeks) significantly increased HDL-C level with independence of the genetic background without elevating TC level.   SQ supplementation can modify HDL composition depending on the presence of Apo E and Apoa1.   SQ supplementation can elicit an antioxidant action by decreasing oxidative stress level in lipoprotein fractions.
Study 5 Kumar et al. 2016 [52]	Hyperlipidemia	Animal study   Obtained from Wako Pure Chemicals, Ltd., Osaka, JP	Obese/diabetic model KK-Ay mice (male, four weeks age)   The mice were randomly divided into control and experimental groups (n = 7).	The groups were as follows: control fed with fat diet and test diet fed with fat diet contained 2% SQ.   At the end of the experiment (4 weeks), all animals were sacrificed to collect blood and other organs.   Measurement involved i)fatty acid profile and ii)TG, cholesterol levels in serum, liver, and epididymal white adipose tissue (WAT).	There was a significant decrease in the liver and epididymal WAT TG of SQ fed experimental groups as compared to control (p < 0.05).   TG levels in serum and cholesterol levels in serum, liver, and epididymal WAT were not significantly different from control(p > 0.05).   There was a significant increment in proportions of the long-chain n-3 fatty acid, DHA, in the squalene-fed group when compared to control (p < 0.05).	SQ influenced the lipid metabolism as seen by the TG levels and fatty acid profiles in the test diet fed to KK-Ay mice.   SQ potentially exhibits a hypotriglyceridemic effect.
Study 6 Smith et al. (2000) [53]	Hyperlipidemia	Animal study   Obtained from Sigma, St. Louis, MO	Thirty-six male adult F1B hamsters (170 to 250 g) During phase three, the animals were randomly assigned to three different diet groups (12 animals each).	This study was divided into three phases: phase 1 (acclimatization): the animals were fed with a normal chow diet; phase 2: the animals were fed with a high-fat diet for four weeks; and phase 3: the animals were randomly assigned to the following three groups for four weeks: i) group 1 continued with the same high-fat diet, ii) group 2 was fed a high-fat diet supplemented with 1% SQ, and iii) group 3 was fed a high-fat diet plus 0.5% β-sitosterol.   At the end of each phase, major lipoproteins, namely chylomicrons, VLDL + IDL, and HDL were isolated and their concentrations were measured.	Cholesterol: During phase 3, both un-supplemented and SQ-supplemented groups (groups 1 and 2) showed significant increases in cholesterol content in VLDL + IDL (p< 0.01) compared with their phase 2 values. There were no significant changes observed in any of the groups on LDL-C and HDL-C levels.   Triglycerides/ HDL and LDL-C ratios: During phase 3, group 1 and group 2 had comparable plasma triglyceride levels. The SQ-supplemented group (Group 2) significantly increased plasma triglyceride and LDL-C/HDL-C ratio in comparison to the level at phase 1.	Under this experimental condition, SQ supplementation at 1% of the total diet did not produce hypocholesterolemic and hypotriglyceridemic effects in the high-fat diet hamsters.
Study 7 Zhang (2002) [44]	Hyperlipidemia	Animal study   Obtained from Sigma, St. Louis, MO	Thirty male Golden Syrian hamsters (95 ± 5 g)   Divided into five groups (n = 6)	Each group was fed with one of the following diets for 4 weeks: 1. high-fat diet (HFD); 2. HFD + 0.05% pure SQ; 3. HFD + 0.1% pure SQ; 4. HFD + 0.5% pure SQ; and 5. HFD + 0.05% SQ-containing shark liver oil (SLO).   At the end of the treatment, blood was sampled for serum lipid measurements. Livers, heart, and adipose tissue were collected for cholesterol measurement.	Elevation in serum TC in all groups supplemented with SQ (groups 2 to 5)   In comparison to the control, serum TC was significantly (p < 0.05) increased in groups 2 and 4. A similar trend was observed for serum TG.   Significant elevation in serum HDL-C in the 0.1% SQ and 0.5% SQ groups but not in the 0.05% SQ group as compared with the control hamsters   No significant differences in serum non-HDL-C were observed among the five groups.   SQ supplemented at 0.5% had significantly increased the cholesterol level in the liver and adipose tissue compared to the control. No differences in the cholesterol levels in the heart were observed among the five groups.	The investigators had concluded that SQ exerts a hypercholesterolemic effect at least in hamsters. Thus, caution must be exercised when SQ is routinely consumed as supplements.
Study 8 Castro et al. 2013 [54]	Hyperlipidemia	Animal study   Amaranth oil (in-house extraction)	Forty-six male Golden Syrian strain hamsters, weanling (approximately 21 days), of sanitary standard type   The animals were randomly divided into five groups.	Following seven days of adaptation, the rats were fed with commercial diet.   Prior to further dietary regimen, six animals were randomly selected and fasted for 8 h before blood collection. The remaining 40 animals were divided into groups according to different diets and were fed for four weeks: i) control was fed a balanced diet containing 20% corn oil as the lipid source; ii) hypercholesterolemic was identical to the control group but contained 12% coconut oil, 8% corn oil, and 0.1% cholesterol as the lipid source; iii) Amaranth oil was identical to the hypercholesterolemic group but substitute corn oil with amaranth oil; and iv) squalene was identical to the hypercholesterolemic group but admixed with SQ in the ratio found in amaranth oil.   At the end of the experiment, the animals were sacrificed for blood and liver collection. The following parameters were included: a) analyses in plasma (TC, TG, and HDL-C) and non-HDL-C (LDL-C + VLDL-C), b) analyses in the liver: liver weight and hepatic cholesterol concentration, c) histological analysis: haematoxylin and eosin, and d) analyses in the feces: cholesterol and bile acids.	There was a significant increase in all lipid profile parameters in the amaranth oil and SQ groups when compared to the control group (p < 0.05).   Fecal excretion of bile acids was significantly greater in the amaranth oil and SQ groups as compared to the control group (p < 0.05).   The liver weight was significantly increased in the SQ group compared to the control group (p < 0.05).	The consumption of amaranth oil and its SQ component did not exert a hypocholesterolemic effect in hamsters fed on a high-fat diet but promoted an increase in fecal excretion of bile acids.

VLDL: very-low-density lipoproteins; HDL: high-density lipoprotein; ROS: reactive oxygen species; IDL: intermediate-density lipoproteins; Apoa1: apolipoprotein a-1;CVD: cardiovascular disease; DHA: docosahexaenoic acid; KK-Ay: a cross between diabetic kk and lethal yellow; BW: body weight; LDL-C: low-density lipoprotein cholesterols; HDL-C: high-density lipoproteins cholesterols; TAG: triacylglycerol; TC: total cholesterol; TG: triglyceride; AS: amaranth SQ; SS: shark liver SQ; F1B: bio F1B; ApoE: apolipoprotein E; Apoa4: apolipoprotein A4.