ALMOND-CITRUS PEEL FORMULATED SHORTBREAD MODULATES PLASMA

LIPID PROFILE AND OXIDATIVE STATUS IN HYPERLIPIDEMIC HYPERTENSIVE

RATS

Ayokunle Ademosun a*, Opeyemi Ojueromia a,b, Ganiyu Oboh a

a
Functional Foods and Nutraceuticals Unit, Department of Biochemistry, Federal University of

Technology, Akure, Ondo State, Nigeria.

b
Functional Foods and Nutraceuticals Unit, Department of Pure and Applied Sciences, Precious

Cornerstone University, Ibadan, Oyo State, Nigeria.

*Corresponding author

Ayokunle Ademosun

Email: ojueromiopeyemi@pcu.edu.ng

ABSTRACT

Background: Dietary sources of functional foods and nutraceuticals have demonstrated significant

potentials in the treatment of hypercholesterolemia and hypertension. Almond nuts (Terminalia

catappa) and Orange (Citrus sinensis) peels have a long folklore history in the treatment of

hypercholesterolemia and hypertension. This study aim to investigate the effects of almond and

citrus peel formulated shortbread on lipid profile, oxidative stress and antioxidant parameters in

high fat diet (HFD)/Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME)- induced

hyperlipidemic-hypertensive rats.
Methods: The experimental animals were distributed into eight groups and were orally

administered L-NAME (40mg/kg) and HFD. They were fed with varying amounts of almond and

citrus peel formulated shortbread [0.2% citrus peel, 50% almond and combination of almond

(50%)-citrus peel (0.2%). Thereafter, the effect of almond and citrus peel formulated shortbread on

plasma lipid profile and antioxidant indices of the experimental rats were determined.

Results: The results showed a significant decrease in atherogenic index, total cholesterol,

triglycerides, low density lipoprotein (LDL), malondialdehyde (MDA) and reactive oxygen species

in the rats fed with almond and citrus peel formulated shortbread compared to the HFD/L-NAME-

induced rats. In addition, the almond and citrus peel formulated shortbread groups showed

significant increase in the level of HDL cholesterol and activities of the superoxide dismutase and

catalase of the induced rats.

Conclusions: The results showed that almond and citrus peel formulated shortbread improved

plasma lipid profile and antioxidant status in HFD/L-NAME- induced rats. Intriguingly, the

almond (50%)-citrus (0.2%) formulated shortbread had the best antioxidative and

antihyperlipidemic effects.

Keywords: Almond-Citrus Shortbread; Functional Foods; Hyperlipidemia; Hypertension.

 CHAPTER ONE

1.0 INRTODUCTION

Hyperlipidemia and hypertension the most prevalent metabolic disorders affecting millions of

people worldwide, and the principal risk factos for cardiovascular disease (CVD) [1].

Hyperlipidemia is a principal precursor to many cardiovascular, cerebrovascular, and peripheral

vascular diseases. Approximately 110 million people are affected by hyperlipidemia with an

estimated 4.4 million deaths globally [2]. Hyperlipidemia is characterized by increased lipid

level in the blood, particularly LDL-cholesterol leading to the development of atherosclerosis, a

condition characterized by plaque buildup in the arteries [3] while, hypertension refers to high

blood pressure above 140/90mmHg [4], which can damage blood vessels and trigger the risk of

cardiac diseases. These two risk factors often coexist and lipid profile assays are typically used to

assess the severity of hyperlipidemia and its associated risk factors, including high levels of low-

density lipoprotein (LDL) cholesterol and triglycerides, and low levels of high-density

lipoprotein (HDL) cholesterol [5]. Other oxidative biomarkers, including ROS, and

malondialdehyde (MDA), provide insights into the underlying pathophysiology of

hyperlipidemia [6]. Oxidative stress occurs when there is an imbalance between ROS production

and the body's antioxidant defense system [7]. Hyperlipidemia has been shown to increase

oxidative stress levels in the body, leading to the development of various cardiovascular diseases

[8]. The low-density lipoprotein (LDL) is commonly referred to as bad cholesterol. Elevated

levels of LDL-cholesterol, which are observed in hyperlipidemia, can lead to the formation of

oxidized LDL-cholesterol particles. These particles are highly reactive and can induce the

production of ROS, which can damage the endothelial cells lining the blood vessels. This
damage can result in the development of atherosclerosis, narrowing the blood vessels and

restricting blood circulation to vital organs such as the heart and brain [9].

In addition to using medication as the first line of defense in treating hyperlipidemia

which are often accompanied with deleterious side effect, some other options that can be

considered effective involves the intake of medicinal plants and lifestyle modification. Due to the

increasing prevalence of hyperlipidemia, as well as the drug resistance and side-effects of current

anti-hyperlipidemic drug, attention has been shifted towards medicinal foods.

The therapy of coronary heart disorders, hypertension, and hyperlipidemia is aided by

the availability of medicinal plants rich in antioxidants and phytochemical components [10].

Almond (Terminaliacatappa) is a type of tree nut that are commonly eaten as a snack or used as

an ingredient in cooking and baking. They are native to the Middle East and have been cultivated

for millennia. Orange peel (Citrus sinensis) contains higher concentration of flavonoids and

phytonutrients than its inner flesh. It is usually cultivated in tropical region because of its edible,

ornamental and medicinal purpose [11]. For this reason, orange peels are credited with a number

of pharmacological properties. Almond nut and orange peel are commonly used as nutraceuticals

in folkloric medicine for the treatment of diverse cardiovascular and degenerative diseases,

although there is limited evidence on the scientific basis for its use. This study sought to evaluate

the antioxidative and lipid lowering abilities of almond-citrus peel formulated short bread in

HFD/L-NAME-induced hyperlipidemic hypertensive rats.

 CHAPTER TWO

3.0 MATERIALS AND METHODS

3.1 Materials

3.1.1 Sample collection

Oranges, purpose flour, greek yoghurt, almond nuts, aspartame and skimmed milk were bought

at Akure main market, Nigeria. The commercial shortbread was procured from a minimart at

FUT, Akure.

3.1.2 Chemicals

Chemicals were procured from Sigma-Aldrich Chemical Co. The chemical reagents used for the

assay were of standard analytical grades, while the lipid profile was determined using kit

reagents and distilled water.

3.2 Methods

3.2.1 Sample preparation

Orange peels were air dried for some days and blended to powered form, almond nut was milled,

measured and mixed together with skimmed milk, water, aspartame and other materials in

different proportions .The dough was then cut into shapes and baked in an electric oven.
 Table 1:Composition of almond and citrus peel fortified shortbread

Shortbread All- Almond Greek Skimmed Orange Aspartame (g)

Purpose
Constituent Flour (g) Yoghurt (g) Milk (g) Peel (g)

Flour (g)

Plain Short Bread 100.2 - 50 10 - 0.3

Citrus Peel Short Bread 100 - 50 10 0.2 0.3

(0.2%)

Almond Short Bread (50%) 50.2 50 50 10 - 0.3

Almond (50%)-Citrus Peel 50 50 50 10 0.2 0.3

Short (0.2%) Bread

 COMMERCIAL

SHORTBREAD

PLAIN

SHORTBREAD

CITRUS PEEL
(0.2%)
SHORTBREAD

ALMOND(50%)
SHORTBREAD

Alm
ALMOND(50%)-
CITRUS PEEL
(0.2%)
SHORTBREAD

Plate 1: Images showing commercial, plain and almond-citrus peel fortified shortbread
3.2.2Experimental Design

Mature male wistar albino rats (weight: 180 - 200 g) were gotten from Animal house,

Department of Biochemistry, Federal University of Technology, Akure, Nigeria, Maintenance of

the wistar rats was done at room temperature. They were allocated into 8 groups and

acclimatized for 2 weeks with access to diet and water. The experimental rats orally administered

L-NAME (40 mg/kg body weight) were fed with high fat diet for 21 days to induce

hyperlipidemia in rats. The HFD/L-NAME hypertensive- hyperlipidemic rats were fed with short

bread supplemented with almond and citrus peel at varying dietary inclusions throughout the

experiment.

Group 1 – NORMAL CONTROL

Group 2- HIGH FAT DIET + L-NAME (40 mg/kg)

Group 3 - HIGH FAT DIET + L-NAME + LISINOPRIL (0.14mg/kg) + STATIN (0.2 mg/kg)

Group 4 - HIGH FAT DIET + L-NAME + COMMERCIAL SHORT BREAD

Group 5 - HIGH FAT DIET + L-NAME + PLAIN ALL-PURPOSE FLOUR SHORT BREAD (APF)

Group 6 – HIGH FAT DIET + L-NAME + APF + CITRUS PEEL SHORT BREAD (0.2%)

Group 7 – HIGH FAT DIET + L-NAME + APF + ALMOND FLOUR SHORT BREAD (50%)

Group 8 - HIGH FAT DIET + L-NAME + APF + ALMOND FLOUR (50%) + CITRUS PEEL

SHORT BREAD (0.2%)

3.2.2.1 Preparation of Tissue Homogenate

After treatment, animals were sacrificed by cervical dislocation after an overnight fast. The

blood was taken from the heart into ethylene-diaminetetraacetic acid (EDTA)-containing tubes

and centrifuged at 10,000 × g for 15 minutes

3.3 Biochemical Assays

3.3.1 Determination of Lipid Profile

The rats’ lipid profile ( total cholesterol, high density lipoprotein (HDL), low- density lipoprotein

(LDL) and triglycerides) were derived using Randox assay kits.

3.3.2 Determination of Malondialdehyde (MDA) level

Malondialdehyde (MDA) content in the blood was assayed by the method of Ohkawa et al [12].

Three hundred microlitre of tissue homogenate, three hundred microlitre of 8.1% SDS (Sodium

dodecyl sulfate), Five hundred microlitre of acetic acid/HCl (pH 3.2) and 0.8% TBA were added,

and the mixture was incubated at 100 °C for 1 h. Thereafter, the thiobarbituric acid reactive

species (TBARS) produced was read at 532 nm.

3.3.3 Determination of Superoxide dismutase activity

The method of Alia et al [13] to evaluate the inhibition of autoxidation of epinephrine at pH 10.2

and 30°C superoxide dismutase (SOD) activity determination.

3.3.4 Determination of Catalase (CAT) activity

The activity was determined in a reaction involving heart homogenate, H 2O2 plus buffer (pH 7).

The experiment procedure was halted with potassium dichromate prepared with acetic acid. The

H2O2 consumed was read at 260nm and expressed as mol/mg protein [14].
3.3.5 Determination of Reactive Oxygen Species (ROS) Concentration

The ROS level was measured utilizing the method of Hayashi et al. [15]. Fifty microlitre of

tissue homogenate and 1400 µL of CH3COONa buffer (0.1 M, pH 4.8) was transferred into a

beaker and a thousand microlitre of reagent mixture (containing 6 mg/mL N,N, diethyl-

paraphenylenediamine and 4.37 µM Ferrous sulphate dissolve in CH3COONa buffer) was added.

The absorbance was read at 505 nm.

3.3. Protein determination

The protein content was analyzed by the Coomassie blue method utilizing serum albumin as

standard [16].

3.4 Statistical Analysis

Data are expressed as mean ± standard error of the mean (SEM). All the data were analyzed

using GraphPad statistical package, version 8.0 while significance level, which was accepted at p

<0.05 after Tukey's post hoc test using one- way ANOVA.
Figure 1: Effect of almond-citrus peel formulated shortbread on plasmaatherogenic index
(AI) in HFD/L-NAME induced hyperlipidemic-hypertensive rats.

Results are expressed as mean ± standard error of the mean (SEM). *Values are significantly p <
0.05 different from HFD + L-NAME-induced group. #Values are significantly p < 0.05 different
from HFD + L-NAME + Statin + Lisinopril.

Control; HFD+L-NAME = High fat diet + Nω-Nitro-L-arginine-methyl ester; HFD+L-

NAME+STATIN+LIS = High fat diet + Nω-Nitro-L-arginine-methyl ester + statin + lisinopril;
HFD+L-NAME+COMBREAD = High fat diet + Nω-Nitro-L-arginine-methyl ester +
commercial shortbread; HFD+L-NAME+APF = High fat diet + Nω-Nitro-L-arginine-methyl
ester + all-purpose flour; HFD+L-NAME+APF+CITRUS(0.2%) = High fat diet + Nω-Nitro-L-
arginine-methyl ester + all-purpose flour + citrus peel (0.2%) ; HFD + L-NAME + Almond
(50%) = High fat diet + Nω-Nitro-L-arginine-methyl ester + almond flour (50%) ; HFD + L-
NAME +Almond (50%) + citrus peel (0.2%) = High fat diet + Nω-Nitro-L-arginine-methyl ester
+ almond (50%) + citrus peel (0.2%).

Figure 2: Effect of almond-citrus peel formulated shortbread on plasma triglyceride

activity in L-NAME induced hyperlipidemic-hypertensiverats.

Results are expressed as mean ± standard error of the mean (SEM). *Values are significantlyP<
0.05 different fromHFD+L-NAME-induced group.#Values are significantlyP< 0.05 different
fromHFD+L-NAME+Lisinopril + Statin group
Figure 3: Effect of almond-citrus peel formulated shortbread on plasma total cholesterol
(TC) activity in L-NAME induced hyperlipidemic-hypertensiverats.

Results are expressed as mean ± standard error of the mean (SEM). *Values are significantlyP<
0.05 different fromHFD+L-NAME-induced group.#Values are significantlyP< 0.05 different
fromHFD+L-NAME+Lisinopril + Statin group
Figure 4: Effect of almond-citrus peel formulated shortbread on plasma low density
lipoprotein (LDL) level inL-NAME induced hyperlipidemic-hypertensiverats.

Results are expressed as mean ± standard error of the mean (SEM). *Values are significantlyP<
0.05 different fromHFD+L-NAME-induced group.#Values are significantlyP< 0.05 different
fromHFD+L-NAME+Lisinopril + Statin group
Figure 5: Effect of almond-citrus peel formulated shortbread on plasma high density
lipoprotein(HDL) level in L-NAME induced hyperlipidemic-hypertensive rats.

Results are expressed as mean ± standard error of the mean (SEM). *Values are significantlyP<
0.05 different fromHFD+L-NAME-induced group.#Values are significantlyP< 0.05 different
fromHFD+L-NAME+Lisinopril + Statin group
Figure 6: Effect of almond-citrus peel formulated shortbread on plasmamalondialdehyde
(MDA) activity in L-NAME induced hyperlipidemic-hypertensiverats.

Results are expressed as mean ± standard error of the mean (SEM). *Values are significantlyP<
0.05 different fromHFD+L-NAME-induced group.#Values are significantlyP< 0.05 different
fromHFD+L-NAME+Lisinopril + Statin group.
Figure 7: Effect of almond-citrus peel formulated shortbread on plasma reactive oxygen
species(ROS) activity in L-NAME induced hyperlipidemic-hypertensiverats.

Results are expressed as mean ± standard error of the mean (SEM). *Values are significantlyP<
0.05 different fromHFD+L-NAME-induced group.#Values are significantlyP< 0.05 different
fromHFD+L-NAME+Lisinopril + Statin group
Figure 8: Effect of almond-citrus peel formulated shortbread on plasmasuperoxide
dismutase (SOD) activity in L-NAME induced hyperlipidemic-hypertensiverats.

Results are expressed as mean ± standard error of the mean (SEM). *Values are significantlyP<
0.05 different fromHFD+L-NAME-induced group.#Values are significantlyP< 0.05 different
fromHFD+L-NAME+Lisinopril + Statin group
Figure 9: Effect of almond-citrus peel formulated shortbread on plasmacatalase (CAT)
activity in L-NAME induced hyperlipidemic-hypertensiverats.

Results are expressed as mean ± standard error of the mean (SEM). *Values are significantlyP<
0.05 different fromHFD+L-NAME-induced group.#Values are significantlyP< 0.05 different
fromHFD+L-NAME+Lisinopril + Statin group
Figure 10: Effect of almond-citrus peel formulated shortbread on plasma glutathione

peroxidase(GPx) activity in L-NAME induced hyperlipidemic-hypertensiverats.

Results are expressed as mean ± standard error of the mean (SEM). *Values are significantlyP<

0.05 different fromHFD+L-NAME-induced group.#Values are significantlyP< 0.05 different

fromHFD+L-NAME+Lisinopril + Statin group

 CHAPTER FIVE

3.0 RESULTS AND DISCUSSION

Hyperlipidemia represents a medical term for abnormally high levels of fats (lipids) in the blood,

which include cholesterol and triglycerides. Hyperlipidemia is a known culprit in the incidence

of cardiovascular diseases [17]. Excessive dietary fat intake can negatively affect lipid

metabolism leading to hyperlipidemia [18] and increase the level of angiotensin II, a factor in

hypertension development.

Statins are standard drugs commonly used to treat hyperlipidemia and to halt the progression of

atherosclerosis-related problems like heart attacks and stroke. These medications are relatively

expensive and often accompanied with attendant side effects including rhabdomyolysis,

diarrhoea and dizziness [19]. However, the use of medicinal plants/foods to regulate lipid or

cholesterol levels has grown enormously and has gained global acceptance in the nutraceutical

sector. Citrus and almond plants, which are rich in phenolics and flavonoids have been reported

to possess pharmacological potentials like anti-stress and cardoprotective properties [20].

Remarkably, results from this study revealed no significant (P< 0.05) difference between the

group treated with the standard drugs (simvastatin and lisinopril) and the group treated with

almond (50%)-citrus peel (0.2%) formulated shortbread.

Atherogenic index of plasma constitutes triglycerides and high-density lipoprotein cholesterol

[21]. It is used to measure blood lipid levels and also frequently utilized as a good biomarker for

dyslipidemia and cardiovascular disorders. In this study, a noticeable elevation was observed in

the plasma atherogenic index, triglycerides and total cholesterol level of the untreated

hyperlipidemic-hypertensive rats (Fig.1-3). However, the treatment with the almond-citrus peel
shortbread reversed the effect. The elevated level of total cholesterol and triglyceride is

connected to cardiovascular abnormalities and could be as a result of high flux of fatty acids and

abnormal elimination of low density lipoprotein (LDL) from the plasma [22]. In physiological

state, the plasma LDL consists of triglycerides and cholesterol esters with an outer layer made up

of free cholesterol, apolipoprotein B (ApoB) and phospholipids that transport hydrophobic

cholesterol in circulation [23]. The LDL oxidation triggers lipid peroxidation process principally

involving the phospholipid molecules during oxidative state. Individuals with higher LDL levels

have a higher probability of developing cardiovascular illnesses, according to clinical and

epidemiological studies [24]. In order to transport cholesterol back to the liver, high density

lipoprotein (HDL) encourages the absorption of cholesterol from a variety of peripheral tissues,

including the artery wall. The HDL may provide protection by preventing the oxidation of LDL

and counteracting the atherogenic effects of oxidized LDL, as well as by reversing cholesterol

transport [25]. This study demonstrated reduced LDL and elevated HDL level in the rats treated

with the almond-citrus peel formulated shortbread in the hyperlipidemic-hypertensive rats

(Fig.4-5). However, the groups treated with almond (50%)-citrus peel (0.2%) formulated

shortbread significantly (P< 0.05) increased HDL and lowered LDL level than the individual

effect of almond (50%) and citrus peel (0.2%) shortbread.

The oxidative damage to lipids, proteins, deoxyribonucleic acid (DNA), and tiny biomolecules is

caused by reactive oxygen species (ROS). According to recent research, hyperlipidemia under

conditions of oxidative stress is a risk factor for the development of atherosclerosis and aberrant

lipid metabolism, and oxidative stress is the primary mechanism triggering cardiovascular

disorders [26]. Hyperlipidemia may result in the elevated production of ROS, which exert their

cytotoxic effect by triggering malondialdehyde formation (Makhdoumi et al., 2020).

Malondialdehyde (MDA) is a prime index for assessing lipid peroxidation. The result showed

significant elevation in the MDA and ROS level of the untreated hyperlipidemic-hypertensive

rats (Fig.6 -7). However, the treatment with shortbread formulated with the individual effect of

almond (50%) and citrus (0.2%) as well as its combination [almond (50%)-citrus (0.2%)]

reduced MDA and ROS levels in the hyperlipidemic-hypertensive rats. Intriguingly, the almond

(50%) -citrus peel (0.2%) shortbread elicited the best anti-oxidative effect. In this study, the

observed rise in plasma MDA and ROS level in the untreated HFD/L-NAME-inducedrats may

indicate deficiency of antioxidant defense system and/or induction of oxidative stress in

hyperlipidemic and hypertensive conditions. The treatment with almond-citrus peel shortbread

may further suggest potential protection of the rats against the progression of oxidative stress-

induced pathologies.

Citrus peels and almond nuts have been reported to be rich in natural antioxidants including

polyphenols and flavonoids [27]. Antioxidants prevent the impairment of biological tissues

caused by oxidative stress. Endogenous antioxidants may not be adequate to stop damage, thus

dietary antioxidants may be required to prevent diseases caused by free radicals. [28]. These

findings gave rise to the theory that dietary antioxidants could boost cardioprotection and

improve blood lipid profiles by reducing oxidative stress and suppressing macromolecule

oxidation. [29]. The results presented in Fig. 8 – 9 illustrate the effects of shortbread formulated

with citrus peel and almond on plasma antioxidant enzyme in HFD/L-NAME induced rats. The

results showed a marked (P< 0.05) reduction in the activities of superoxide dismutase (SOD) and

catalase (CAT) in the plasma of untreated hyperlipidemic-hypertensive rats. However, the

treatment with the standard drugs (lisinopril& simvastatin) as well as the varying dietary

inclusions of almond (50%), citrus peel (0.2%) andalmond (50%)-citrus peel (0.2%) significantly
 elevated SOD and CAT in the HFD/L-NAME- induced hyperlipidemic-hypertensive rats.

Interestingly, shortbread with the combination of almond (50%) and citrus peel (0.2%) showed

the highest SOD and CAT activities in hyperlipidemic-hypertensive rats. The initial line of

cellular defense against oxidative damage is provided by the SOD and CAT, which are involved

in the elimination of superoxide anions and hydrogen peroxide [30]. Studies have shown that

SOD and CAT are comparatively stable enzymes that may become inactive in the presence of

extreme oxidative stress. Thus, an imbalance between the antioxidant and oxidant systems may

result from insufficient detoxification of these ROS by antioxidant enzymes. The decreased SOD

and CAT activity may potentially be due to ROS-induced enzyme inactivation, which damage

biological tissues. [31].

CONCLUSION

This study revealed that almond-citrus peel enriched short bread improved plasma lipid profile and

antioxidant status in the hyperlipidemic-hypertensive rats. Intriguingly, the almond (50%)-citrus

(0.2%) formulated shortbread had the best antioxidative and antihyperlipidemic effect. The dietary

supplementation with almond nuts and citrus peels could be a promising strategy in the prevention

and/or management of hyperlipidemia and hypertension.

Acknowledgements

The authors would like to appreciate Functional Foods and Nutraceutical Unit, Department of

Biochemistry for the facilities provided during the present study.

Availability of data and materials

The data will be made available on request

Authors’ contributions

AA conceived the research work ; AO and OO supervised the project and gave the required tips

and scientific instructions. The article was prepared by OO. All authors read and approved the

final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

No funding was obtained for this study

REFERENCES

1. Alloubani A, Nimer R, Samara R. Relationship between hyperlipidemia, cardiovascular

disease and stroke: a systematic review. Current Cardiology Reviews. 2021; 17(6), 52-66.

2. CDC (2022) How and When to Have Your Cholesterol Checked | cdc. gov. Centers for

Disease Control and Prevention. Published April 15, 2021.

https://www.cdc.gov/cholesterol/checked.htm.

3. Feng X, Zhang L, Xu S, Shen AZ. (2020). ATP-citrate lyase (ACLY) in lipid

metabolism and atherosclerosis: An updated review. Prog lipid Res. 2020; 77;101006.
4. Morales-Villegas EC. Yarleque C, Almeida ML. (2023). Management of hypertension

and dyslipidemia in Mexico: evidence, gaps, and approach. Archivos de cardiología de

México. 2023; 93(1): 77-87.

5. Crismaru I, Pantea Stoian A, Bratun OG, Gaman MA, Stanescu AMA, Bacalbasa N,

Diaconu CC. (2020). Low-density lipoprotein cholesterol lowering treatment: the current

approach. Lipids in health and disease. 2021; 19:1-10.

6. Oguntibeju OO, Aboua Y, Goboza M. Vindoline—a natural product from Catharanthus

roseus reduces hyperlipidemia and renal pathophysiology in experimental type 2

diabetes. Biomed. 2019: 7(3), 59.

7. Khutami C, Sumiwi SA, Khairul Ikram NK, Muchtaridi M. The effects of antioxidants

from natural products on obesity, dyslipidemia, diabetes and their molecular signaling

mechanism. Intl J Mol Sci.2022; 23(4): 2056.

8. Lorenzon dos Santos J, Schaan de Quadros A, Weschenfelder C, Bueno Garofallo S,

Marcadenti A. . Oxidative stress biomarkers, nut-related antioxidants, and cardiovascular

disease. Nutr.2020; 12(3):682.

9. Miao J, Zang X, Cui X, Zhang J. (2020). Autophagy, hyperlipidemia, and

atherosclerosis. Autophagy: Biology and Diseases: Clinl Sci 2020; 237-264.

10. Chukwu E, Osuocha KU, Iwueke AV. (2020). Phytochemical profiling, body weight

effect and anti-hypercholesterolemia potentials of Cnidoscolus aconitifolius leaf extracts

in male albino rat. J Pharmacog Phytother.2020; 12(2):19-27.

11. Memariani Z, Farzaei MH, Ali A, Momtaz S. (2020). Nutritional and bioactive

characterization of unexplored food rich in phytonutrients. In Phytonutrients in Food

2020;157-175). Woodhead Publishing.

12. Okhawa H, Ohishi N, Yagi K. Anal. Biochem. 1979; 95: 351

13. Alia M, Horcajo C, Bravo L, Goya L. Effect of grape antioxidant dietary fiber on the

total antioxidant capacity and the activity of liver antioxidant enzymes induced by

thermal oxidation of dietary lipids in rats. Nutrition. 2003; 10: 797-805

14. Shina JL. Catalase. In Methods of enzymatic analysis. Academic Press. 1972; 885-894

15. Hayashi I, Morishita Y, Imai, K, Nakamura M, Nakachi K, Hayashi T. High-

throughput spectrophotometric assay of reactive oxygen species in serum. Mutation

research/genetic toxicology and environmental mutagenesis. 2007 1 :55-61.

16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities

of protein utilizing the principle of protein-dye binding. Analytical biochemistry. 1976;

7;72(1-2)

17. Dubey A, Dash SL, Kumari M, Patel S, Singh S, Agarwal S. A Comprehensive Review

on Recent Progress in Invivo and Invitro Models for Hyperlipidemia Studies. Pakistan

Heart J. 2023; 56(1):286-297.

18. Singh A. Hyperlipidemia in cardiovascular health and digestion. In Nutrition and

Functional Foods in Boosting Digestion, Metabolism and Immune Health 2022; 141-

150. Academic Press.

19. Ji E, Lee S. Antibody-based therapeutics for atherosclerosis and cardiovascular

diseases. Intl J Mol Sci, 2021; 22(11):5770.

20. Mahmoud YT, Khalil FA, Abd El-Rhman A. Cardioprotective Effects of Cape

Gooseberry (Physalis Peruvian) andor Loquat (Eriobotrya Japonica) Juices Through

Decreasing Serum B-Type Natriuretic Peptides (BNPNT-proBNP) in High Fat-High

Cholesterol Feeding Rats.

21. Ramchoun M, Khouya T, Harnafi H, Alem C, Benlyas M, Simmet T, Amrani S. Effect

of polyphenol, flavonoid, and saponin fractions from Thymus atlanticus on acute and

chronic hyperlipidemia in mice. Future J. Pharm Sci. 2020: 6(1), 1-9.

22. Arnold SV, De Lemos JA, Rosenson RS, Ballantyne CM, Liu Y, Mues KE, GOULD

Investigators. Use of guideline-recommended risk reduction strategies among patients

with diabetes and atherosclerotic cardiovascular disease: insights from Getting to an

Improved Understanding of Low-Density Lipoprotein Cholesterol and Dyslipidemia

Management (GOULD). Circ, 2019; 140(7): 618-620.

23. Su L, Mittal R, Ramgobin D, Jain R, Jain R. Current management guidelines on

hyperlipidemia: the silent killer. Journal of lipids. 2021.

24. Stein R, Ferrari F, Scolari F. Genetics, dyslipidemia, and cardiovascular disease: new

insights. Curr Cardiol Reports. 2019; 21: 1-12.

25. Mardi P, Abdi F, Ehsani A, Seif E, Djalalinia S, Heshmati J, Qorbani M. Is non-high-

density lipoprotein associated with metabolic syndrome? A systematic review and meta-

analysis. Front Endocrinol 2022; 13: 957136.

26. Guo L, Xiao J, Liu H, Liu H. Selenium nanoparticles alleviate hyperlipidemia and

vascular injury in ApoE-deficient mice by regulating cholesterol metabolism and

reducing oxidative stress. Metallomics, 2020; 12(2):204-217.

27. Ling Y, Shi Z, Yang X, Cai Z, Wang L, Wu X, Jiang, J. Hypolipidemic effect of pure

total flavonoids from peel o f Citrus (PTFC) on hamsters of hyperlipidemia and its

potential mechanism. Experimental gerontology, 2020; 130: 110-786.

28. Adedapo KS, Adepoju S, Olusanya TO. Effects of Selected Antioxidants on

Atherosclerosis in Hyperlipidemic Wistar Rats. atherosclerosis (ECCA). 2019; 34.

29. Olorunnisola OS, Adegbola PI, Ajilore BS, Akintola OA, Fadahunsi OS. The role of

poly-herbal extract in sodium chloride-induced oxidative stress and hyperlipidemia in

male wistar rats. Medicine. 2021; 8(6):25.

30. Ramos LPA, Justino AB, Tavernelli N, Saraiva AL, Franco RR, de Souza AV,

Espindola FS. (2021). Antioxidant compounds from Annona crassiflora fruit peel reduce

lipid levels and oxidative damage and maintain the glutathione defense in hepatic tissue

of Triton WR-1339-induced hyperlipidemic mice. Biomed & Pharmacother.

2021; 142:112049.

31. Byrne NJ, Rajasekaran NS, Abel ED, Bugger H. Therapeutic potential of targeting

oxidative stress in diabetic cardiomyopathy. Free Rad Biol Med. 2021; 169: 317-342.