Introduction
Reactive Oxygen Species (ROS) are the amounts of reactive molecules and free radicals derived from oxygen in a molecule (
Peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α is a transcription coactivator that regulates the genes involved in energy metabolism. It is the main regulator of mitochondrial biogenesis ( Liang &Ward, 2006). PGC-1α stimulates mitochondrial biogenesis and encourages the remodeling of muscle tissue to a fiber-type composition that is metabolically more oxidative and less glycolytic in nature, and it participates in the regulation of both carbohydrate and lipid metabolism ( Puigserver & Spiegelman, 2003; S. Yang et al., 2020).
The ability of cell defense against ROS has been associated with aging and contributes to the increased oxidative stress state. This condition can disturb the enzyme activity, especially through the reversible oxidative reaction at the thiol functional group at the side chain of the enzyme structures ( Birben et al., 2012). This can lead to the alteration of biomolecule structure and integrity, and enzyme dysfunction ( Freitas et al., 2016). As a result, insulin resistance and Type 2 Diabetes can development ( Facchini et al., 2001; Meigs et al., 2003). Additionally, the effect of aging on changes in liver mass can increase serum Low-Density Lipoprotein (LDL)-cholesterol level, due to the hepatocytes cell death caused by oxidative stress ( Anantharaju et al., 2002; Miller, 1984). Hence, effective control of ROS levels is essential. The aging population tends to have a higher prevalence of chronic disease, thus there is a demand for health-improving foods ( Park, 2013). The consumption and production of high-antioxidant as functional foods in recent years are popular due to their capability of reducing Reactive Oxygen Species (ROS), as well as having an impact on several aging and chronic related diseases ( Park, 2013; Park et al., 2004). However, there are some challenges that are associated with the utilization of functional food. For example, specific functional foods need to be consumed in high concentrations in order to be biologically effective, therefore, this would require the nutritional facts such as the daily dose of the bioactive compound in each serving size to be determined ( Kang et al., 2011). Preliminary studies are needed to determine which bioactive compound is the most beneficial, and what is the quantitative-activity relationship between the bioactive compounds contained in functional foods.
Sea grapes (
Methods
This
Collection and preparation of plant material
Fresh sea grapes (
Preparation of sea grapes extracts
Crude powder (one kg) was macerated in 96% ethanol for 72 hours with each extraction performed in triplicates, which resulted in 34% yield. The crude extracts were filtered by Whatman 41 filter paper. The total filtrate was concentrated and evaporated at 40°C with a rotary evaporator RV 8 IKA under reduced pressure (100 millibar) for 90 minutes, and evaporated in an 40°C oven to produce a thick extract. The extract was stored in a refrigerator at 10°C until used in the study.
Animal handling and ethical approval
All experimental rats were kept on standardized free access of feed and
CFED production
Carbohydrates fat-enriched diets (CFED) is a standard mouse food that comes with 1% colic acid, 2% pure cholesterol powder, 20% fat (animal source/pork oil), and 2% corn oil. Additional components are subtly added to the standard CFED and homogenized into a dough with the addition of 1000mL of aqua dest. Small pellets are cut and left to dry at room temperature in sterile conditions. CFED is prepared weekly and stored at 4°C until used to reduce oxidation. CFED consists of carbohydrate (43.57%), coarse protein (12.38%), coarse fiber (4.73%), coarse fat (3.17%), cholesterol (2%), colic acid (1%), animal fat (20%), corn oil (2%), total ash (4.3%), and moisture (6.85%). Compared to a normal diet (standard dry pellet) that contains 58.1% carbohydrates, 16.51% coarse protein, and 0% animal fat, all the other components such as corn oil, cholesterol, and folic acid, were not significantly changed. CFED production guideline was carried out as previously described (Harb et al., 2018).
Sea grapes extract administration scheme
Wistar albino rats were randomly distributed into four groups of ten animals each. Group A served as control (received standard dry pellet diet). Rats in group B were fed on CFED for 4 weeks. Rats in groups C and D were fed on CFED and were given 150 and 450 mg/kg Body Weight (BW) of sea grapes extract, respectively, for 4 weeks. CFED and extract of sea grapes were administered by oral gavage.
Sample collection
Throughout the experiment, all the efforts were made to minimize the pain and distress of the experimental animals. For this purpose after four weeks of extract treatment, rats were kept fasted overnight and given euthanasia under ketamine anesthesia. 2.5 mL of blood samples were collected from the tail and kept in dry and clean tubes without addition of anticoagulants (Tiger-top tube), to allow clotting at room temperature. The samples were then centrifuged for 20 minutes at 3000 rpm. Finally, the sera were collected for the blood glucose, total cholesterol, and PGC-1α analysis.
Biomedical analysis of blood sample
Blood glucose and cholesterol levels were assayed using COBAS Integra ® 400 plus analyzer (Roche) ( See underlying data) ( Nurkolis, 2021). Samples were washed with Phosphate Buffered Saline (PBS, pH 7.4) 1% until the liquid runs clear. The samples were centrifuged at 3000 rpm for 20 minutes to obtain pellets and supernatant. The supernatant is taken for the PGC-1α examination ( See underlying data) ( Nurkolis, 2021). The concentration of PGC-1α was measured by using mouse PGC-1α ELISA Kit (Sunlong Biotech Co., Ltd, # MBS288117).
Data management and analysis
The data were statistically analyzed with the use of the MANOVA/Multivariate ANOVA test. The Levene’s test was used to determine which posthoc tests should be conducted. In cases where the Levene’s test p-value was <0.05 Games-Howell test (equal variances not assumed), and for p-value >0.05 Bonferroni test (equal variances assumed) was used. Statistical analyses were performed by using SPPS 26.0 for the Windows version.
Results
Table 1.
Statistical interpretations based on homogeneity test.
F | df1 | df2 | P-value | ||
---|---|---|---|---|---|
Glucose | Mean | 10.495 | 3 | 36 | .000 * |
Median | 7.105 | 3 | 36 | .001 * | |
Median (adjusted df) | 7.105 | 3 | 19.296 | .002 * | |
Trimmed mean | 10.205 | 3 | 36 | .000 * | |
Cholesterol | Mean | 1.957 | 3 | 36 | .138 |
Median | 1.741 | 3 | 36 | .176 | |
Median (adjusted df) | 1.741 | 3 | 23.800 | .186 | |
Trimmed mean | 1.853 | 3 | 36 | .155 | |
PGC-1α | Mean | 9.042 | 3 | 36 | .000 * |
Median | 6.290 | 3 | 36 | .002 * | |
Median (adjusted df) | 6.290 | 3 | 29.106 | .002 * | |
Based on trimmed mean | 8.938 | 3 | 36 | .000 * |
F, Levene's Test; DF, Degrees of Freedom.
*
Represents p-value <0.05, CI:95%.
The Levene's Homogeneity test shows that the p-value for glucose and PGC-1α are <0.05, therefore equal variance cannot be assumed, while equal variances can be assumed for cholesterol as the p-value is >0.05.
The results indicate that blood glucose significantly increased in group B, compared to group A (p < 0.05) ( Table 2). Blood glucose significantly decreased in both groups C and D (p < 0.05). The effect of sea grapes administration as much as 150 mg/kg BW is more effective than the sea grapes 450 mg/kg BW, in significantly decreasing blood glucose in rats (p < 0.05).
Table 2.
The low dose of sea grapes is more effective in significantly reducing blood glucose.
Diet | Mean | P-value | |
---|---|---|---|
Group A | CFED | −15.3880 | .000 * |
CFED + Sea grapes 150 mg/kgBW | 5.9500 | .000 * | |
CFED + Sea grapes 450 mg/kgBW | 2.8900 | .001 * | |
Group B | Control | 15.3880 | .000 * |
CFED + Sea grapes 150 mg/kgBW | 21.3380 | .000 * | |
CFED + Sea grapes 450 mg/kgBW | 18.2780 | .000 * | |
Group C | Control | −5.9500 | .000 * |
CFED | −21.3380 | .000 * | |
CFED + Sea grapes 450 mg/kgBW | −3.0600 | .003 * | |
Group D | Control | −2.8900 | .001 * |
CFED | −18.2780 | .000 * | |
CFED + Sea grapes 150 mg/kgBW | 3.0600 | .003 * |
*
Represents p-value <0.05, CI: 95%.
As expected the rats in group B had significantly increased blood cholesterol levels compared to group A (p < 0.05). In both groups A and B (p < 0.05), blood cholesterol significantly decreased in rats given CFED + sea grapes extract 150 mg/kg BW, and CFED treatment + sea grapes extract 450 mg/kg BW. There was no significant difference between the CFED treatment group + 150 mg/kg BW sea grapes extract, and the CFED treatment group + 450 mg/kg BW sea grapes extract, in reducing blood cholesterol (high dose of the extract did not result in significant effects (p > 0.05)).
Group B had a significantly decreased PGC-1α serum concentration. PGC-1α serum concentrations significantly increased in group C, as well in group D, compared to groups A and B. The effect of sea grapes administration as much as 150 mg/kg BW is more effective than that of sea grapes 450 mg/kg BW, in the significant increase of PGC-1α serum in rats.
Discussion
This study showed that the supplementation of sea grapes extract managed to lower blood glucose and serum cholesterol significantly in rats that were given cholesterol- and fat-enriched diets ( Figure 1). Although compared to the control group, rats that were given cholesterol- and fat-enriched diets with sea grapes extract had lower levels of blood cholesterol and blood glucose.
Figure 1.
Effects of sea grapes extract on subject rats.
The Results of this study indicate that sea grapes have the capability of reducing blood glucose levels ( Table 2). Similarly, Aroyehun et al., have shown that sea grapes have antidiabetic activity ( Qudus B Aroyehun et al., 2020). The plasma analysis in this study has also indicated that the sea grapes treated group had a significant decrease (p < 0.05) in their blood glucose levels compared to the untreated diabetic group ( Qudus B Aroyehun et al., 2020). Sea grapes extract-treated group demonstrated similar efficacy in lowering blood glucose as Metformin ( Qudus B Aroyehun et al., 2020), hence, sea grapes may have an hypoglycaemic effect. A hyperglycaemic state may induce oxidative stress that could be detrimental to insulin-sensitive tissues such as the liver, which may cause damage to the organ (Bugianesi 2005; Manna 2010; Palsamy 2010).
This study showed that sea grapes reduce hyperlipidemia in rats, however this is not in line with the findings by Aroyehun et al., ( Qudus B Aroyehun et al., 2020), which states that sea grapes extract has little to no effect on the cholesterol level of induced diabetic rats. In addition, the effect of lower doses of the extract (150 mg/kg BW) was better in lowering blood cholesterol than higher doses of sea grapes extract (450 mg/kg BW) ( Figure 1, Table 3). This can be due to the saturated fatty acids content, especially palmitic acid, which dominates the composition of fatty acids, comprising 80% of the total fat in sea grapes ( Qudus B Aroyehun et al., 2020). Studies have shown that palmitate acid may raise total cholesterol levels, specifically LDL-cholesterol levels ( Clandinin et al. 2000; Mensink, 2013).
Table 3.
Both doses of sea grapes extract significantly reduce blood cholesterol.
Diet | Mean | P-value | |
---|---|---|---|
Group A | CFED | −22.1100 | .000 * |
CFED + Sea grapes 150 mg/kgBW | 12.0600 | .000 * | |
CFED + Sea grapes 450 mg/kgBW | 8.0200 | .001 * | |
Group B | Control | 22.1100 | .000 * |
CFED + Sea grapes 150 mg/kgBW | 34.1700 | .000 * | |
CFED + Sea grapes 450 mg/kgBW | 30.1300 | .000 * | |
Group C | Control | −12.0600 | .000 * |
CFED | −34.1700 | .000 * | |
CFED + Sea grapes 450 mg/kgBW | −4.0400 | .222 | |
Group D | Control | −8.0200 | .001 * |
CFED | −30.1300 | .000 * | |
CFED + Sea grapes 150 mg/kgBW | 3.0600 | .003 * |
*
Represents p-value <0.05, CI: 95%.
Levels of PGC-1α in rats significantly decreased after being given a CFED diet compared to the control group ( Figure 1, Table 4). However, PGC-1α levels increased significantly in rats given sea grapes extract, even when compared to the control group. This suggests that PGC-1α, which is one of the major elements in mitochondrial biogenesis, is enhanced by the sea grapes extract. Perhaps the content of flavonoids as well as phenols in sea grapes extract can cause this effect. One study has shown that flavonoid supplementation increases the performance in endurance activities via an increase in expression of PGC-1α as the “master regulator” of biogenesis and skeletal muscle angiogenesis ( Khani et al. 2017). In addition, other studies have also shown that antioxidant compounds can upregulate PGC-1α target genes, which not only play a role in preventing oxidative damage, but also reduce mitochondrial ROS levels, ensure mitochondrial integrity during cell differentiation (Beldelli et al. 2014), as well as avoiding the cytotoxic effects of ROS accumulation ( St-Pierre et al. 2006).
Table 4.
The low dose of sea grapes is more effective in significantly increasing PGC-1α.
Diet | Mean | P-value | |
---|---|---|---|
Group A | CFED | 20.9200 | .000 * |
CFED + Sea grapes 150 mg/kgBW | −19.3500 | .000 * | |
CFED + Sea grapes 450 mg/kgBW | −14.4200 | .000 * | |
Group B | Control | −20.9200 | .000 * |
CFED + Sea grapes 150 mg/kgBW | −40.2700 | .000 * | |
CFED + Sea grapes 450 mg/kgBW | −35.3400 | .000 * | |
Group C | control | 19.3500 | .000 * |
CFED | 40.2700 | .000 * | |
CFED + Sea grapes 450 mg/kgBW | 4.9300 | .000 * | |
Group D | control | 14.4200 | .000 * |
CFED | 35.3400 | .000 * | |
CFED + Sea grapes 150 mg/kgBW | −4.9300 | .000 * |
*
Represents p-value <0.05, CI: 95%.
Conclusion
Sea grapes extract is proven to improve blood glucose levels, total cholesterol, and PGC-1α in rats fed with cholesterol- and fat-enriched diets. The results of this study can be used as a reference for clinical trials to further research the beneficial effects of sea grapes for human consumption. However, it is necessary to do the same research with parameters other than blood sugar, cholesterol and PGC-1a, to expand its metabolic scope.
Data availability
Underlying data
Harvard dataverse: Sea grapes extract effect on blood glucose level (BGL), total cholesterol (TC), and serum PGC-1α concentrations.
DOI: https://doi.org/10.7910/DVN/8IKREA ( Nurkolis, 2021).
The project contains the following underlying data:
• Raw data for the sea grapes extract effect on blood glucose level (BGL), total cholesterol (TC), and serum PGC-1α concentrations.
Reporting guidelines
Harvard Dataverse: Arrive checklist for Sea grapes extract with blood glucose, total cholesterol, and PGC-1α in rats fed on cholesterol- and fat-enriched diet.
https://doi.org/10.7910/DVN/NXF0IW ( Nurkolis et al., 2021).
Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).
Author contributions
M.K and F.N. collated study ideas, designed and experiment, analyzed data, and compiled manuscripts. N.A.T, N. R, N. S, H.K.P, D.S.W and N. M analyzed and interpreted the data and critically revised the manuscript. F. N and F.M.I conducted experiments, analyzed biochemistry, and critically revised the manuscript. N. M, M.R.B, R. R, P.S.A and K.E.K.M, implemented experimental protocols, assisted in statistical analysis, interpreted data, and critically revised manuscripts. All writers read and approve the final manuscript.
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Abstract
Background: Sea grapes or Caulerpa racemosa have a lot of phytochemical content, especially unsaturated fatty acids that are beneficial for health. This study aims to evaluate the effects of sea grapes extract on blood glucose levels, total cholesterol-, and Peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α in male Wistar rats, which were given per-oral (p.o.) cholesterol- and carbohydrates fat-enriched diets (CFED). Methods: Forty male Wistar albino rats weighing between 200 – 250 g were used for this study. Animals were randomly distributed into four groups of ten animals each. Group A served as control (received standard dry pellet diet). Rats in group B were fed on CFED for 4 weeks. Groups C and D were fed on CFED and were administered 150 and 450 mg/kg of sea grapes extract (p.o.), respectively. Results: Group C rats indicated a blood glucose reduction and an increase in PGC-1α serum, in comparison to group D (p<0.05). There were no significant differences between group C and D in blood cholesterol reduction (high dose of the extract did not have significant effects) (p=0.222), and both groups had the same effect in lowering total cholesterol in rats. Conclusion: Sea grapes extract is proven to improve blood glucose, total cholesterol, and PGC-1α levels in rats fed with CFED.
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Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer