Anti-atherogenic effect of flaxseed (Linum usitatissimum) administration with a high cholesterol diet and its effect on hyperlipidemia in female Wistar rats.

Background: Cardiovascular disease (CVD) persists to be the world's leading reason for mortality and morbidity. The major type of CVD is coronary artery disease (CAD). CAD is the obstruction of coronary arteries frequently instigated by arteriosclerosis (plaque buildup in the coronary artery, which hampers blood flow to the myocardial muscles). This research was conducted to examine the effects of oral intake of flaxseed (Linum usitatissimum) powder on hyperlipidemia and atherosclerosis (induced through high-fat diet) in female Wistar rats. The pharmacological effects of flaxseed are diverse. It has an antiarrhythmic effect, anti-arthritic effect, antilipidemic, atherosclerosis, anti ‐ inflammatory, antioxidant activity effect on the body. Methodology: A total of 32 female Wistar rats (200 g) were divided equally into four groups. Group I (n=8) rats kept as control and given normal rat chow diet, Group II rats kept as positive control for induction of hypercholesterolemia and atherosclerosis by addition of saturated edible fat to the standard diet (high cholesterol diet - HCD), Group III rats fed with flaxseed powder at 7.5 g/kg of rat/day in the standard rat chow diet and kept as flaxseed control, and Group IV rats supplemented with flaxseed at 7.5 g/kg of rat/day along with HCD and maintained for 15 days. Results: Group II rats showed higher lipid profile and glucose level. Flaxseed supplementation, along with a high cholesterol diet, drastically restored the serum levels of triglyceride (TG), decreasing frequency of CAD. Histologic investigations showed preserved myocardial walls with minor changes in flaxseed treated rats, whereas increased lymphoid aggregation with inflammation in high fat + flaxseed treated. Conclusion: The biochemical assays and histological results showed the moderate cardio-protective activity of flaxseed administration for 15 days along with a high-fat diet was found to moderately reduce the CVD risk in rats.


Introduction
Coronary artery disease is a disorder of coronary arteries which is chronic and multifactorial, develop silently, with no symptoms till the plaque restricts the blood flow 1 . World Health Organization (WHO) estimated 16.7 million deaths are caused annually worldwide due to cardiovascular disease 2 . According to research, in Pakistan, CVD is the largest non-communicable killer 1 .
Eicosanoids originated from ω-3-F.A, in flaxseed principally progresses heart function by decreasing cholesterol level 9,10 . Due to the wide use of herbal medicine and its therapeutic effects, there is growing attention and a need to assess the mechanisms of action of herbal products carefully.
This rising understanding of herbal products attracts attention to holding fewer side effects accompanied by multiple medicinal properties. Therefore, this research intended to scientifically demonstrate the cardioprotective nature of orally administered flaxseed powder against hyperlipidemic induced arteriosclerosis and coronary artery disease in female Wistar rats.

Plant Material
Linum usitatissimum Flaxseed were bought from the local marketplace of Karachi, Pakistan. The seeds were rinsed and dried overnight. The dried seed was crushed into powder form with the help of an electronic crusher. The grind was kept in a clean, airtight container and kept at room temperature.

Animals Housing
Female Wistar rat (Rattus norvegicus) of 15-20 weeks old and with a mean bodyweight of 150-200 g were bought from the International Center for Chemical and Biological Sciences, University of Karachi. Rats were acclimatized for 1 week inside the animal house of the Department of Physiology (University of Karachi) before the research was conducted. Rats were kept in a well ventilated and temperature-controlled room 25-28°C and 14/10 h light/dark cycles, with an unlimited diet approach (rat chow diet) and water in their cages. Body weights and other physical conditions were carefully examined throughout the study.

Ethical Guideline
This study was conducted according to the standards issued by the National Institute of Health.
Study Design 32 rats were distributed into four experimental groups as follows: Group-I: Rats received a normal rat chow diet throughout the experimental period. Group-II: Received high-fat diet (saturated fat/100 g of daily diet) for 15 days. Group-III: treated with flaxseed powder 7.5 g/kg of rat/day in the rat chow diet for 15 days. Group-IV: Received flaxseed powder 7.5 g/kg per day with a high-fat diet for 15 days. On the 16th day, all animals were sacrificed. The cardiac piercing technique was utilized, and heparin-coated tubes were used for acquiring plasma for analysis; serum was also set apart. The heart was removed and made free of connective tissues and blood, followed by desiccation and weighing. Lastly, the tissue was stored in a freezer at -70 ºC.

Biochemical Analysis
Plasma TC, TG, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) was estimated using Global chemical reagent package.

Histopathological
Heart tissue was fixed and submerged using formalin and then turned in paraffin block, respectively. Sections of thickness four µm were stained with Hematoxylin/Eosin stain and examined under the microscope. Histopathological changes in tissue sections were ranked with a scale from 0 to 4 (Zero means no distinguishable, 1-slight localized impairment, 2-mild localized impairment, 3-moderate localized impairment and 4-sever according to the seriousness of structural alternations.

Statistical Analysis
The results are shown as mean ± SEM (Standard error mean). All four groups were analyzed for statistically significant variations using one-way ANOVA, and significance was known at p<0.05, p<0.01 & p<0.005. Tukey's HSD test and Dunnett's test were utilized to assess tested groups' results with controls and to minimalize the occurrence of type I error.

Results
The mean body weight of group I (control group) of Wistar rats on a normal rat chow diet was 129.33± 8.491 g. Group II fed with a high-fat diet for 15 days has non-significant (p<0.05; p=0.00) average weight compared to Group I because the animals gain weight and, hence, the average body weight of Group II was 212.16±5.51 g.
Group III was fed with flaxseed powder of 7.5 g/kg of rat/day in the standard rat chow diet and kept as flaxseed control has average body weight 229.25±16.13 g. In comparison, the bodyweight of Group IV was 197±3.32 g, which was significant (p>0.05, p=0.08) when compared to group III.  Histological findings in the current study are according to the biochemical findings. Scoring of the heart's morphological conclusions are explained and reviewed in Control, Hyperlipidemic, Flaxseed treated and Flaxseed + high fat treated rats (Table 3). Any altered cardiomyocytic histological changes were not present in the control group's heart tissues ( Figure 1).
Group III (Flaxseed treated) hear tissue sections from group III represent increased inflammation and lymphocytic aggregation in myocardial walls. Rats who received only flaxseed with rat chew diet displayed preserved cardiac wall histology (Figure 3). Group IV (Flaxseed + high-fat diet) this tissue sections from group IV displayed increased lymphoid aggregation with inflammation. Flaxseed controls these changes in this group ( Figure 4).

Discussion
Functional foods are considered superior in treating numerous chronic diseases, primarily due to their decreased side effects. Linum Usitatissimum is a rich source of oil, protein and dietary fiber in addition to an excellent resource of the phytochemical antioxidant, lignin 6 . Flaxseed is rich in Alpha-linolenic acid and linoleic acid 6 . Throughout the experimental phase, no atypical clinical symptoms were witnessed in Groups II and IV animals during the research period. Overweight and lethargy were seen in Group II, fed with a highfat diet. Mild obesity was detected in Group IV, in which flaxseed powder was administered with a high-fat diet. Research denoted that an HFD raised the levels of serum cholesterol, which is coherent with former findings 11,12 .
In this research, we observed that Linum Usitatissimum reduce triglyceride more effectively than total cholesterol. Hokanson and Austin reported that raised plasma TG levels were related to increased frequency of coronary artery diseases 13 . Elevated triglyceride levels attribute the deposition of small dense LDL that initiates atherosclerosis in group II 14  Anila and Vijayalakshmi found out that phytochemical examination of linseed displays the existence of flavonoids and phenolics. They are known to be potent antioxidants and control numerous enzyme systems' actions due to their interaction with several biomolecules. In reality, flavonoids and polyphenols display various pharmacological activities, containing hypolipidemic and anti-atherogenic effect 18 . Therefore, the hypolipidemic action of oral admiration of Linum Usitatissimum maybe credits to flavonoid in it along with interdependent action Secoisolariciresinol diglucoside 19 .

Conclusion
This study has highlighted the bioactivities of flaxseed administered for 15 days in experimental models of hypercholesterinemia. We hypothesized that whole flaxseeds would recover cardiovascular changes by balancing the level of proatherogenic parameters. Significant changes might be acquired with prolonged administration of such doses of flaxseed. Regular consumption of flaxseed can be used to limit primary cases of coronary artery diseases; however, more detailed experimental studies are suggested for its use in diagnosed patients.

Conflicts of Interest
None.