In vitro and in vivo hypolipidemic properties of the aqueous extract of Spirulina platensis, cultivated in colored flasks under artificial illumination

Algal cultures

Pure culture of Spirulina platensis was obtained from the Microbiology Department, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Egypt (Hamouda, Sorour & Yehheia, 2016).

Culture maintenance

The blue green alga S. platensis was sustained on Zarrouk’s medium (Zarrouk, 1966; Belay, 1997) having the subsequent constituents Table 1:

The effect of colored flasks on cell dry weight

Red, blue, green and colorless Erlenmeyer flasks containing Zarrouk’s medium were used for culturing S. platensis. All flasks including 100 mL of selected media under aerobic conditions, were autoclaved for 20 min at 121 °C, then incubated at 26 ± 2 °C with incessant illumination by cool white fluorescent with light intensity of 2,500 lux (35 µmol m⁻² s⁻¹), after that left to grow with two times daily shaking to avoid algal cell clumping and adherence of algal cells to the containers. Cultures were gathered after 2 weeks by centrifugation at 5,000 rpm (Centurion Scientific LTD Model 1020 series) for 10 min and washed well with distilled water (Hamouda, Sorour & Yehheia, 2016).

The algal biomass was evaluated by measuring its dry weight. All samples were filtered using filter paper (pore size 8 mm), washed with distilled water, and dried at 60–65 °C till constant weights (Nigam et al., 2007).

Pigments estimation

A known volume of S. platensis culture was centrifuged at 8,000 rpm for 10 min, and then S. platensis pellets were treated with the same volume of 90% acetone, kept in water bath for 30 min at 55 °C, and followed by centrifugation once again at 8,000 rpm for 10 min. The color of pellets must be white to guarantee complete extraction of pigments, after that supernatant was collected and then the absorbance was measured at three different wavelengths 663, 645 and 480 nm. Calculations were made according to the formula devised by the method of Jeffrey & Humphrey (1975) for chlorophyl a, and Ridley (1977) for total carotenoid estimation.

Chlorophyll a = [12.7 A₆₆₃ − 2.69 A₆₄₅] × vol. of extraction/weight of the sample

where A₆₆₃ is the absorbance at 663 nm and A₆₄₅ is the absorbance at 645 nm.

Total carotenoid (mg/g) = 4 × A₄₈₀ × vol. of extraction/weight of the sample

where A₄₈₀ is the optical density at 480 nm and 4 is the correction factor.

Preparation of algal extracts and in vitro anti-cholesterol assay

Algal extracts were prepared according to Hamouda et al. (2017). Different amounts of dried algal biomass (50, 100, 150 and 200 mg) were boiled in 10 mL distilled water for 30 min, cooled and centrifuged at 1,000 rpm for 5 min, after that the supernatants were taken as the algae extracts.

One hundred µL of each algae extracts was added to 100 µL of cholesterol standard (The cholesterol 200 mg was dissolved in 100 mL of phosphate buffer, pH 7.0 and 1% of Triton X-100 surfactant), mixed and incubated for different periods (15, 30, 45, 60 min) at 37 °C. The cholesterol concentration was measured using enzymatic colorimetric method according to Richmond (1973), using cholesterol kit “Cholesterol–Liquizyme (Elitech, Puteaux, France)”. The contents were incubated for 10 min at room temperature and the absorbance of sample and standard were read at wavelength 500 nm in a UV-Vis spectrophotometer against reagent blank. Anti-cholesterol activity of the extract was calculated using the following equation: $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\mathrm{\%})=\mathrm{Standard\; conc.}\mathrm{-}\mathrm{Sample\; conc./Standard\; conc.}\times 100$$

Extraction and determination of fatty acid composition

One g of dry powder of S. platensis was extracted twice with 20 mL methanol 99.5%, and was stirred with magnetic stirrer for 15 min; then the supernatant was collected by filtration. All supernatants were collected as a bold fraction and the solvent was evaporated using rotary evaporator. The methyl esters can be attained by transmethylation of the lipids by refluxing them for 90 min with methanol-benzene-sulfuric acid (20:10:1), respectively (Harborne, 1973; Mendham et al., 2000). The solution was concentrated to two thirds of its volume, water was then added for washing till free from acidity as indicating by litmus paper indicator, dried over anhydrous sodium sulfate and filtered, and the chemical composition was performed using Trace GC-ISQ Q mass spectrometer (Thermo Scientific, Austin, TX, USA) according to the method described by El-Dougdoug et al. (2018).

Feeding study

Histopathological examination

Liver and heart specimens were sliced, and pieces were preserved in 10% formalin for proper fixation. These tissues were processed and embedded in paraffin wax. Sections of 4–5 microns in thickness were cut and stained with hematoxylin and eosin. All the stained sections of the tissues were examined under microscope for circulatory disturbances, inflammation, degenerations, apoptosis, necrosis, and any other pathological changes, according to the method of (Suvarna, Layton & Bancroft, 2013).

Statistical analyses

All data were subjected to analysis of variance (ANOVA). Three replicates of each item were analyzed and the main values as well as the SD were given. Significance of the variable mean differences was determined using Duncan’s multiple range tests (p ≤ 0.05). All analyses were carried out using SPSS 16 software (Sokal & Rohlf, 1995).

Estimation of algal growth, chlorophyl a and carotenoid

Results in Table 3 A represent the effect of cultivation of blue-green algae S. platensis in flasks with different colors (colorless, red, green and blue) on its cell dry weight, chlorophyl a and total carotenoid contents.

The highest cell dry weight of S. platensis (3.40 g/L) was observed using the colorless flasks that was followed by (2.60 and 2.18) g/L using the red and blue colored flasks, respectively. On the other hand, the green-colored flasks gave the lowest cell dry weight with an average of 2.00 g/L.

Spirulina platensis cultivated in colorless flasks demonstrated the highest chlorophyl a content 17.85 mg/g (1.78%) based on dry weight, while S. platensis cultivated in green-colored flasks had the lowest chlorophyl a content 10.50 mg/g (1.05%). On the other hand, the highest contents of carotenoid 61.88 mg/g (6.19%) was detected in dried cells of cultivated S. platensis in colorless flasks, followed by 47.32 mg/g (4.73%) using the red-colored flasks and 39.68 mg/g (3.97%) using the blue-colored flasks, respectively. While the green-colored flasks, gave the lowest carotenoid content with an average of 36.40 mg/g (3.64%). There are variations in pigment content in all experimental trials, which may be associated with color of flasks.

In vitro cholesterol-lowering activity of Spirulina platensis cultivated in colored flasks

To investigate the potential cholesterol-lowering effect of S. platensis; we carried out a screening on the cholesterol-lowering abilities of different hot water extracts of dried S. platensis obtained from different colored glass flasks. An anti-cholesterol assay was performed and the results revealed the potential activity of all extracts of algae in a dose-dependent manner.

Figure 1A shows the percentages of the cholesterol reducing activity of water extract of S. platensis cultivated in colorless flasks as a control treatment. The highest cholesterol reduction percentage (76.01%) was observed using the hot water extract of S. platensis at a concentration of 15 mg/mL after 60 min of incubation that was followed by 5 mg/mL (74.64%) followed by 20 mg/mL (72.27%) after incubation for time intervals of 60 and 30 min, respectively. On the other hand, the concentration of 5 mg/mL gave the lowest cholesterol reduction percentage with an average of 61.85% value (p > 0.05) after a period of 15 min. No significant differences were observed between the treatments, furthermore the S. platensis concentration and incubation time had no significant effect on cholesterol-lowering levels. Figure 1B illustrates the percentages of the cholesterol reduction of S. platensis obtained from blue flasks. No significant differences were observed between the treatments, moreover the S. platensis concentration and incubation time had no significant effect on cholesterol-lowering levels; and the reduction rate ranged between 45.5% and 48.4%. Figure 1C illustrates the percentages of the cholesterol reduction at different periods of time and different concentrations of dry weight of S. platensis obtained from green flasks. The highest cholesterol reduction percentage (52.8%) was observed using the hot-water extract of S. platensis at a concentration of 15 mg/mL after 60 min of incubation; on the other hand, the concentration of 20 mg/mL gave the lowest cholesterol reduction percentage with an average of 40.9% value (p < 0.05) after a period of 15 min. No significant differences were observed between the treatments, furthermore the S. platensis concentration and incubation time had no significant effect on cholesterol-lowering levels. In the case of red flasks (Fig. 1D), the highest cholesterol reduction percentage (52.9%) was observed using the hot-water extract of S. platensis at a concentration of 20 mg/mL after 60 min of incubation.

GC-MS analysis of Spirulina platensis

Methanolic extract of S. platensis cultivated in colorless flasks was analyzed by GC-MS for determining of its volatile components based on their retention time and peak area (Table 4; Fig. 2). The GC-MS analysis showed 15 bioactive compounds were identified and grouped according to their chemical structures. The major volatile components contained were isochiapin B (3.81%), oxiraneundecanoic acid, 3-pentyl (2.32%), dimethoxy glycerol docosyl ether (2.1%), 9 hexadecenoic acid, eicosyl ester (1.69%), hexamethylcyclotrisiloxane (1.23%), 9-octadecenoic acid (Z)-, phenylmethyl ester (0.69), oleic safflower oil (0.66%), palmitic acid (0.51%), 10,13-octadecadiynoic acid (0.56%), 9-octadecenoic acid, (2-phenyl-1,3-dioxolan-4-yl) methyl ester, cis- (0.46%), 7,10-pentadecadiynoic acid (0.44%), propanedioic acid dimethyl ester (0.31%), palmitic acid (2-phenyl-1,3-dioxolan-4-yl) (0.35%).

The presence of antioxidant compounds such as mono and polyunsaturated fatty acids in the microalgae Spirulina can be the cause of the distinguishing characteristics of Spirulina on the reduce of serum lipid levels. The GC-MS analysis of the S. platensis methanolic extract shows fatty acids that have also been reported to have some anti-cholesteremic activity, especially eicosenoic acid and high-oleic safflower oil (sunflower oil is mainly triglycerides, typically derived from the fatty acids linoleic acid “poly-unsaturated omega-6” and “oleic acid” “mono-unsaturated omega-9” with differing concentrations). Hexadecanoic acid (palmitic acid) ethyl ester, exists naturally in butter, cheese, milk, and meat, as well as cocoa butter, soybean oil, and sunflower oil; also sodium palmitate is authorized as a natural additive in organic products (US Soil Association standard 50.5.3).

Evaluation of body weight and lipid profile

The body weights of mice that received S. platensis water extracts are listed in Table 5. It showed that the mice fed on high fats diet increased in weight after 5 weeks of treatment and the mice injected with (15 mg/mL) oral gavage with S. platensis only decreased in weight from 29.0 g at the beginning of experiment to 24.7 g by 14.9%.

The mice fed on high fat diet with the same concentration of S. platensis decreased from 31.0 g at the beginning to 23.7 g at the end of the experiment by 23.7%, while the mice fed on high fat diet with S. platensis in concentration of 7.5 mg/mL + atorvastatin (5 mg/Kg body weight) decreased from 28.0 g at the beginning to 23.5 g at the end of the experiment by 16.1%.

Figure 3 showed that mice fed on high fat diets showed significant increase (p < 0.05) in total cholesterol (TC), triglycerides (TG) and LDL and significant decrease (p < 0.05) in HDL compared to those fed on normal diet. After 5 weeks of mice treatment with S. platensis only, serum TC, TG and LDL levels were reduced significantly (p < 0.05), while HDL showed significant increase (p < 0.05) as compared to before treatment.

Hypercholesterolemic mice treated with S. platensis (7.5 mg/mL) + atorvastatin (5 mg/Kg) showed significant decrease (p < 0.05) in serum TC, TG and LDL levels (89.9, 59.4 and 22.5 mg/dL, respectively) and significant increase in the HDL cholesterol level 55.5 mg/dL (p < 0.05) compare with control. Also it is clear that the HDL contents were greater in all treatments of S. platensis groups especially S. platensis only group since it reached 58.8 mg/dL.

Evaluation of liver and kidney functions

Figure 4 showed that mice fed on high fat diets showed significant increase (p < 0.05) in AST and ALT liver enzymes (108.4, 85.5 U/L, respectively) compared to animals fed on normal diet and S. platensis diet only (92.0, 79.2 U/L, respectively). On the other hand liver enzymes significant decreased (p < 0.05) in hypercholesterolemic mice which treated with S. platensis only or in combination with atorvastatin.

Toxic renal effects are normally manifested with increases in the level of serum urea. The recorded values of renal parameters (urea and creatinine) indicate significant reduction, for the all trials of S. platensis with mice (Fig. 5). Consequently, group III showed an increase in the level of renal values, while groups IV, V and VI showed reduction in the level of renal values. On the other hand, groups I and II were considered normal cases.

Histological study

Normal saline (negative control, group I): liver showed normal hepatic parenchyma with preserved lobules, cords, sinusoids, bile canaliculi, portal area and stromal structures. The kupffer cells were prominent. Heart sections revealed normal cardiomyocytes with a few hyaline degeneration in some of them (Fig. 6). S. platensis only (group II): liver showed normal hepatic parenchyma with preserved lobules, cords, sinusoids, bile canaliculi, portal area and stromal structures with prominent kupffur cells. Heart sections revealed mild interstitial edema and myocardial degeneration (Fig. 7). The mice fed on high fat diets (positive control, group III): liver sections showed wide spread centrolobular and periportal micro and macrosteatosis beside portal round cells aggregation. Heart sections revealed mild congestion of the coronary blood vessels and intermuscular capillaries. Moderate number of cardiomyocytes showed macrosteatosis. Interstitial edema was also seen (Fig. 8). The mice fed on high fat diets with S. platensis (group IV): liver sections showed wide spread centrolobular and periportal micro and macro steatosis. Mild portal round cells aggregations and prominent kupffer cells were observed. Heart: showed mild congestion of the coronary blood vessels and intermuscular capillaries. Mild to moderate number of cardiomyocytes showed macrosteatosis. Interstitial edema was also seen (Fig. 9). The mice fed on high fat diets with atrovastin 10 mg/Kg instead of S. platensis (group V): liver sections showed congestion of hepatic blood vessels with perivascular leukocytic infiltration mainly neutrophils in addition to extravasated erythrocytes. Examined sections from the heart showed sever congestion of coronary blood vessels, interstitial edema beside steatosis and hyaline degeneration of some cardiomyocytes (Fig. 10). The mice fed on high fat diets with atrovastin 5 mg/Kg and S. platensis 7.5 mg/Kg (group VI): liver sections revealed apparently normal hepatic parenchyma with preserved lobules, cords and sinusoids beside presence some dispersed apoptotic and de-generated cells. Heart sections revealed normal cardiomyocytes with mild interstitial edema and hyaline degeneration in some cells (Fig. 11).

Scope for future work

Microalgae are among the fastest growing photosynthetic organisms giving a higher yield than other crops they have unique properties of CO₂ fixation and release of oxygen. The research findings and trials of this paper can be a fillip for further research on production of low cost Spirulina with superior quality via optimizing physical and chemical conditions of production medium; moreover further clinical studies are needed for confirming some pharmaceutical properties such as anti-diabetic and cancer activities.