INTRODUCTION:

Astaxanthin is a xanthophyll carotenoid.¹ The main biological sources of astaxanthin are crustacea crustacean extracts, the green microalga Haematococcus pluvialis, the yeast Rhodotorula rubra, and the red yeast Phaffia rhodozyma. The aforementioned natural sources cannot compete with the synthetic products available.² Crustacean meals have low levels of astaxanthin and high levels of moisture, ash, and chitin. The algae Haemotococcus pluvialis has a relatively high concentration of astaxanthin, the yeast, has been reported to contain astaxanthin³, but the extremely thick cell wall is a obstacle for the extraction of astaxanthin from Rhodotorula rubra. Some bacterial species (Mycobacterium lacticola) produce astaxanthin only in hydrocarbon medium not on nutrient agar.⁴ have reported astaxanthin in floral parts of Tagetes erecta and Circubita maxima marica, but the seasonal and geographic variations⁵ and the amount of astaxanthin compared to the total mass of the plant is quite small, therefore plants were not considered a suitable source for commercial cultivation.⁶ Animals cannot synthesize astaxanthin and is thus acquired through diet. The mammals and most of the fish are capable of converting dietary carotenoids into astaxanthin. The crustaceans (such as shrimp and some fish species like koi carp) have a limited capacity for converting closely related carotenoids into astaxanthin. However, these are benefited when astaxanthin is directly fed to them. Neither mammals can synthesize astaxanthin nor they can convert it into vitamin A as there is no pro-vitamin activity for astaxanthin unlike β carotene.⁷ The production of free radicals like hydroxyl and peroxyl radicals along a highly reactive forms of oxygen like singlet oxygen occurs during normal physiological processes. The various forms of stress can enhance their production (like physiological stress), air pollution, tobacco smoke, chemical exposure or exposure to UV radiation. The phagocytes can generate free radicals to carry out defensive degradation of an invader. These free radicals have the potential to damage DNA, proteins and lipid membranes which may appear in the form of aging, atherogenesis, ischemia-reperfusion injury, infant retinopathy, age-related mucular degeneration and carcinogenesis.⁸ Membranous phospholipids and other lipids are fairly protected by astaxanthin against peroxidation.^9,10 Antioxidant activity of astaxanthin was found more than β-carotene and vitamin E.¹¹ Haematococcus pluvialis, freshwater green microalga is considered a potent producer of astaxanthin (3, 3′-dihydroxy- β β - carotene- 4, 4′-dione).^12-15 In our previous study^16,17 H. pluvialis was cultivated and astaxanthin was extracted from the green alga H. pluvialis. In present investigation, efforts were made to study the application part of biomass and crude astaxanthin.

MATERIALS AND METHODS:

Procurement of culture and cultivation of H. pluvialis:

The Haematococcus pluvialis culture was procured from the Culture Collection of Algae at the University of Texas, Austin, USA. H. pluvialis culture was grown in the bold basal medium (BBM). The encysted red cells rich in astaxanthin were harvested, dried in the oven at 70^oC.

Extraction of Astaxanthin from H. pluvialis:

10 mg of biomass was homogenized in mortar and pestle in the presence of neutral glass powder (Borosil Glass Works Ltd., India) extracted with acetone and centrifuged 5000rpm 10 minutes. The supernatant was used for the estimation of total astaxanthin. The absorbance of the extracts was determined at 492nm and the amount of the pigment was calculated according to Davies (1976).¹⁸ Acetone was evaporated in the rotatory evaporator and separated by high-pressure thin liquid chromatography.

Ethical statement:

All animal experiments were approved by Institutional Animal Ethics Committee (IAEC) of Pinnacle Biomedical Research Institute (PBRI), Bhopal (Reg. No. 1824/PO/ERe/S/15/CPCSEA). Animal experiments were conducted according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC accreditation).

Animals used for the Study:

Male Albino Wistar rats, weighing 200 ± 20gm were used for the study. Animals were housed in separate cages under controlled conditions with temperature 22 ± 2°C. All animals were given standard diet obtained from Golden Feed, New Delhi) and water regularly. The animals were divided into six groups (Table 1). Group-1 served as normal, group-II served as toxin, group III, VI and V were given a single dose of CCl₄ 2.0g/kg b.w. dissolved in an equal volume of liquid paraffin to produce hepatotoxicity. Administration (orally of standard, biomass, crude extract, and ascorbic acid were started 2 weeks before to CCl₄ treatment. The animals were sacrificed after 24 h of CCl₄treatment, collected liver tissues and blood samples (from the retro-orbital puncture).

Table 1: Grouping of animals for carbon tetrachloride (CCl₄) treatment

Group-1	Control (Untreated group)
Group-2	CCl₄ + normal saline
Group-3	CCl₄ + standard (astaxanthin)
Group-4	CCl₄ + biomass
Group-5	CCl₄ +crude extract
Group-6	CCl₄ + ascorbic Acid

Lipid peroxidation assay:

Lipid peroxidation activity was determined spectrophotometrically at 532nm by the procedure of Ohkawa (1979).¹⁹

Analysis of antioxidant enzymes of liver tissue:

Activity of catalase was measured by the method of Sapakal et al. (2008)²⁰ and superoxide dismutase by the method of Paoletti et al. (1986).²¹ Glutathione’s activity was measured by the procedure of Ellman (1959).²²

Histopathological Studies:

Liver tissues were dissected out and fixed in 10% formalin and embedded in paraffin. Sections were prepared and then stained with hematoxylin and eosin dye and observed under the microscope.

Carbon tetrachloride (CCl₄) Induced Hepatotoxicity:

Carbon tetrachloride (CCl₄) induced hepatotoxicity was performed according to the method of Ranga et al. (2015).²³ Rats were divided into six groups, each group consisted of six animals, (Table 1) Group-1 served as normal, group-II served as toxin, group III, VI and V were given a single dose of CCl₄ 2.0g/kg b.w, dissolved in an equal volume of liquid paraffin to produce free radicals. Administration of standard, biomass, crude extract, and ascorbic acid was started 2 weeks before to CCl₄ treatment. The animals were sacrificed after 24 h of CCl₄ treatment, rats were anesthetized with diethyl ether, serum and liver samples was collected by retro orbital puncture, allowed to clot for 1-2 hour at room temperature and serum was separated by centrifugation at 2500rpm for 15 min and used to determine the activities of SGPT, SGOT, ALP, and total bilirubin.

Statistical Analyses:

All the experimental analyses were done in triplicates. Result values were expressed as mean ± SD and values were evaluated by one-way ANOVA followed by multiple comparison procedures (Bonferroni t-test).

RESULTS AND DISCUSSION:

Cultivation of H. pluvialis:

Haematococcus pluvialis was grown in the bold basal medium for 30 days and the cells were harvested from astaxanthin accumulated red stage (Figure 1).

Figure 1: Haematococcus pluvialis (a) Green stage and red of H.pluvialis (b) Dried biomass

Before proceeding for purification and identification of astaxanthin in acetone extract of Haematococcus pluvialis, astaxanthin extracted with acetone was separated by high-pressure thin layer chromatography to get a piece of first hand information. The R_f values for the bands were 0.21 which were corresponded in accordance with the standard astaxanthin R_f value (Figure 2).

Figure 2: High pressure thin liquid chromatography pigment profile of astaxanthin extracted from Haematococcus cells: (a and c) acetone extract (b and d) astaxanthin standard.

Impact of biomass and crude extract ascorbic acid on the level of lipid peroxidase, superoxide dismutase, GSH and catalase on normal and intoxicated rats:

In the toxin (CCl₄) treated group, lipid peroxidase damaged was increased by 63% when compared with the normal (untreated) group. Treatments of rats with toxin at 2.0g/kg body weight with astaxanthin standard 100 µg/kg b.w significantly restored the levels of peroxidase by 51.53%, whereas when the rats with toxin were treated with 100µg/kg b.w of H. pluvialis biomass, crude extract and ascorbic acid, the peroxidase was restored from damage induced by toxin by 23.61%, 17.49%, 35.18% respectively when compared with the toxin treated control group (Table 2).

In toxin (CCl₄) treated group the SOD damage was increased by 272% astaxanthin standard significantly restored the SOD by 67.22%, whereas when the rats with toxin were treated with 100µg/kg b.w of H. pluvialis biomass, extract and ascorbic acid, the SOD level was restored from damage by toxin by 42.86%, 24.36%, 29.89% respectively when compared with the toxin treated control group.

In the GSH, astaxanthin standard 100µg/kg b.w significantly restored the levels of (GSH) by 57.95%, however when the rats with toxin were treated with 100 µg/kg b.w of H. pluvialis biomass, extract and ascorbic acid, the level of (GSH) was restored from damage by toxin by 33.92%, 22.91% and 45.58% respectively when compared with the toxin treated control group which elevated the GSH damage by 167%. Similarily in toxin (CCl₄) treated group, catalase damaged was increased by 289% when compared with the normal (untreated) group but astaxanthin standard significantly restored the levels of catalase damage from toxin by 69.41%, and H. pluvialis biomass, extract and ascorbic acid, restored the catalase from damage induced by toxin by 52.7%, 40.49 % and 61.94% respectively when compared with the toxin treated control group.

Effect of H. pluvialis biomass and crude extract on hepatoprotective activity in normal and toxin treated rats:

The hepatoprotective activity of the normal and toxin treated rats was evaluated by measuring the content of SGPT, SGOT, ALP and TB. In the CCl₄ (2.0g/kg body weight) treated group serum glutamate pyruvate transaminase damage increased by 63% when compared with the normal group (untreated) but when rats were treated with CCl₄ combined with a dose of 100µg/kg of standard, H. pluvialis biomass, extract and ascorbic acid, SGPT was restored by 52.8%, 29.57%, 16.0%, and 35% respectively when compared with toxin treated group. In the CCl₄ treated group, SGOT content was increased by 66% but its damage was restored when the rats were treated with CCl₄ combined with a dose of 100µg/kg of standard, H. pluvialis biomass, extract and ascorbic by 53.93%, 40.0%, 33.93% and 48.96%. In the same manner in toxin treated group, the ALP content was increased by 49.59% however when rats were treated with CCl₄ combined with a dose of 100µg/kg of standard, H. pluvialis biomass, extract and ascorbic acid, the ALP activity was prevented from damage by 39%, 26.0%, 18.56% and 35.17% respectively. Similarly, TB (total bilirubin) was restored by 73.33%, 57.94, 50.70 and 67.69% respectively from toxin CCl₄and in the toxin treated group the damaged was increased by 77% (Table 3).

An extensive study has been done previously on β-carotene (pro-vitamin-A) and vitamin E. However, in recent times there has been a shift towards the carotenoids like astaxanthin extracted from H. pluvialis which has additional positive features like potent quenching and anti-lipid peroxidation lacking both in β-carotene and vitamin E (Miki, 1991).¹¹ The liver, being a vital organ to metabolise xenobiotics, is affected by free radicals during the catalytic cycle which deactivates the liver detoxification enzymes. The metabolites formed from carbon tetrachloride toxin, which we used in our present investigation damaged liver. The large amount of free radicals in the toxic group trigger the elevation of lipid peroxidation. This propagates the intracellular cytotoxicity by inducing the peroxidation through the interaction of these free radicals with phospholipid structures that destroys the organ structure (Jaeschke et al., 2012).²⁴The free radical interaction with polysaturated fatty acids of the membrane lipids initiate lipid peroxidation causing oxidative stress and formation of end product malondialdehyde (MDA).²⁵ The carotenoids like astaxanthin, lutein and zeastxanthin reduce the risk of various disorders.²⁶ The astaxanthin is found to be effective for cardiovascular disease, blood pressure probably due to its higher antioxidant activity.²⁷ Likewise, 2, 3, 7, 8-tetra chloridebenzodioxin and methyl nitrosourea induced toxicity in rats was reduced through astaxanthin treatment by stimulating the cellular antioxidants enzymes, preventing lipid peroxidation and protein oxidation.^26,28 In rats fed with carbon tetrachloride, lipid peroxidation in serum and the liver, got significantly reduced by astaxanthin treatment relative to rats fed on a controlled diet.²⁹ The carotenoid lutein isolated from marigold flowers (Targetes erecta) protects the liver damage in hepatotoxic rats induced with toxicity by paracetamol, carbon tetrachloride.³⁰ The astaxanthin from H. pluvialis also shows antihypertensive in rats.³¹ The animal experiments have revealed that astaxanthin at levels well above 120 mg a day in human equivalents³², apparently causing no harm, confirmed by various experiments including acute toxicity mutagenicity, teratogenicity, embryotoxicity and reproductive toxicity.³³ Our present study shows that the biomass and crude extract of H. pluvialis have the ability to provide protection to the toxin-induced damage and also maintained healthy to appropriate enzymes in both serum and liver tissue (Table 2 and 3). Histopathology of liver sections in untreated, toxin treated, biomass, crude extract and ascorbic groups were investigated as is shown in (Figure 3). The untreated group showed normal morphology of hepatic cells with the central vein in the middle of the lobule. In CCl₂ treated group, liver sections revealed several changes such as congestion and change in the cell architecture however the liver section have retained the normal cell architecture with a minor injury in biomass and crude extract groups (Figure 3).

CONCLUSION:

The current results revealed that H. pluvialis biomass and crude extract showed umpteen hepatoprotective and antioxidant activity and thus can be used for various food formulations.

ACKNOWLEDGMENT:

We are thankful for the head, department of post graduate studies and research in biological sciences, Rani Durgavati University, Jabalpur - 482001, (M.P.) India for providing necessary facilities.

CONFLICT OF INTEREST:

We declare that we have no conflict of interest

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Received on 17.04.2020 Modified on 01.06.2020

Research J. Pharm. and Tech. 2021; 14(4):1933-1937.

DOI: 10.52711/0974-360X.2021.00342

Treatment Groups	% Anti-lipid peroxidase	Superoxide dismutase (U/gm wet tissue)	Glutathione (nmol/gm wet tissue)	Catalase (U/gm wet tissue
Control (normal)	26.97±3.688	201.43±26.454	0.99±0.022	43.45±5.691
CCl₄ (control)	74.44±3.987	54.14±6.527	0.37±0.027	11.17±1.073
CCl₄+standard	36.08±2.802*	165.21±12.404*	0.88±0.048	36.52±6.873*
CCl₄ +biomass	56.86±2.520*	94.75±12.148*	0.56±0.018*	23.63±2.889*
CCl₄ + crude extract	61.42±2.447*	77.23±11.959^NS	0.48±0.017*	18.77±1.691*
CCl₄ +ascorbic acid	48.25±4.232*	122.61±10.096*	0.68±0.032*	29.35±2.633*

Treatment Groups	SGPT (IU/L)	SGOT (IU/L)	ALP (IU/L)	TB (mg/dL)
Control (normal)	34.50±2.429	43.17±1.472	103.17±10.167	0.43±0.163
CCl₄ (control)	94.67±3.011	129.17±14.798	204.67±8.214	1.95±0.442
CCl₄+standard	44.67±2.582*	59.50±2.665*	123.17±8.864*	0.52±0.117*
CCl₄ +biomass	66.67±6.282*	77.50±6.565*	151.00±11.331*	0.82±0.075*
CCl₄+ crude extract	79.33±7.501*	85.33±3.502*	166.67±8.091*	0.96±0.209*
CCl₄ +ascorbic acid	59.17±2.927*	68.50±5.753*	132.67±12.612*	0.63±0.163*