Ameliorative Effects of Ethanolic Leaf extract of Diodia sarmentosa on high fat induced cardiac tissue injury in male wistar albino rats

doi:10.21203/rs.3.rs-1742900/v2

Background: Diodia sarmentosa is a native straggling herb found in Nigeria, especially in evergreen forest, bushland, and riverine areas. In folklore medicine, it has been used to treat ailments like diabetes, ulcers, etc. This study investigated the ameliorative effects of ethanolic leaf extract of Diodia sarmentosa on high-fat diet-induced cardiac tissue injury in male albino Wistar rats.

Methods: Thirty (30) male Wistar rats (150g-200g) were divided into five (5) groups namely; Negative control (NC) group which was not induced nor treated, Positive control (PC) was induced but not treated, Low dose extract (LDE) was treated with 250mg/kg of the extract, High dose extract (HDE) was treated with 500mg/kg of the extract and SIM group treated with 5mg/kg of Simvastatin. Cardiac tissue injury was induced by feeding the rats with high-fat diet (Ghee and Coconut oil in the ratio of 3:1) for six (6) weeks. The administration of the treatments (extract and drug) at the appropriate doses started from the 4th week till the 6th week. Parameters like Total protein concentration, Lactate dehydrogenase (LDH) activity, Malondialdehyde level, Superoxide dismutase (SOD) activity, Catalase (CAT) activity, Glutathione S-transferase (GST) activity, and Glutathione peroxidase (GPx) activity were carried out in the cardiac tissue of the test rats to determine the ameliorative effects of Diodia sarmentosa.

Results: Results obtained showed that high-fat diet reduced antioxidant enzymes, protein levels, and caused lipid peroxidation indicating injury in the cardiac tissue of the test rats. However, the extract at high dose significantly reduced (p<0.05) lipid peroxidation and high GST activity in the cardiac tissue caused by high-fat diet. Protein levels and antioxidant enzyme activities in SOD, CAT, GPx, and LDH were all significantly increased (p<0.05), when compared to the positive group. The extract at high dose possesses similar efficacy to the standard drug simvastatin used to compare the efficacy of the extract.

Conclusions: Thee evidence from this study shows Diodia sarmentosa has the potential to improve and reverse the injury in the cardiac tissue caused by high-fat diet administration in the test rats, by reducing lipid peroxidation, and increasing antioxidant enzymes and total protein concentration. This makes the plant a potential new natural product for the treatment of cardiovascular diseases.

General Biochemistry

Clinical Pharmacology

Cardiac & Cardiovascular Systems

Cardiac tissue

Diodia sarmentosa

Antioxidant enzymes

Ameliorative effects

High fat diet

Diets containing fat play a vital role in the energy needs, growth, and healthy state of the body [1]. High consumption of dietary fat on the other hand has been reported to cause various health conditions such as oxidative stress, obesity, dyslipidaemia, and hyperlipidaemia . When these health conditions are not properly managed or treated, they lead to the development of cardiovascular diseases like Hypertension, Stroke, Arteriosclerosis, heart attack, and ultimately death [2,3]. These conditions arise when the high fat intake induces oxidative stress in the body, by blocking the hepatic production of the antioxidant glutathione and triggering the oxidation of fatty acids, leading to series of reactions that can alter the immune system [4].

Oxidative stress arises when the ratio of free radicals generated is more than the level of antioxidants used to neutralize those free radicals [5]. This insufficient level of antioxidants is caused by the overproduction of free radical species (reactive oxygen or nitrogen species) thereby weakening the defense system [6]. An extended weakening of the immune system causes oxidative damage to the cells, tissues, and organs leading to the development of health problems like cancers, coronary heart diseases, high blood pressure, stroke, etc [7]. The body experiences oxidative stress when there is an increase in lipid peroxidation products like Malondialdehyde, and a decline in antioxidant enzymes such as Superoxide dismutase, and Catalase that helps in the defense mechanism of the body [5]. Cardiovascular diseases are characterized by high levels of lipid peroxidation products and deficiency in antioxidants such as Glutathione, Superoxide dismutase, Glutathione peroxidase, Glutathione s-transferase, Catalase, etc [8]. Apart from the consumption of diets containing high fat, other activities like high alcohol consumption, use of drugs (anti-inflammation, anti- analgesic, anti-cancer, and anti-depressants), environmental pollution by pollutants, and pesticides can cause oxidative stress in the system [5].

Diodia Sarmentosa (Sw) also known as Zimbabwe flora or tropical buttonweed is from the family of RUBIACEAE. It is a procumbent perennial herb that grows in open riverine vegetation, bushland, and on rocky places close to rivers [9]. Various authors have reported that Diodia sarmentosa possesses a wide range of pharmacological properties like anti-ulcer, anti-inflammatory and analgesic, anti-diabetic, and anti-cancer potentials against ethyl nitrosamine induced hepatocellular carcinoma albino rats [9-12]. It was also reported to scavenge free radicals and possess antioxidant potentials in high-fat-fed rats [13]. A study by Ezejiofor & Korie [14] reported the antihyperlipidaemic potentials of high-fat-fed rats. This study demonstrated the ameliorative effects of the ethanolic leaf extract of Diodia sarmentosa on cardiac tissue injury caused by high-fat diet in male albino Wistar rats.

Plant materials

Fresh samples of Diodia sarmentosa (Sw) leaves were collected from farmlands and natural vegetation within Ihiagwa environment.

Chemicals and reagents

Analytical grade chemicals and reagents were used for this study.

Experimental animals

Male Wistar albino rats weighing between 150-200g were used for this study. The animals were purchased from the Department of Biochemistry, University of Port Harcourt, Rivers State, Nigeria.

Preparation of plant extract

Fresh leaves of Diodia sarmentosa (Sw) were air-dried at room temperature and then ground into a fine powder using laboratory mortar and pestle. These leaves now in the fine powder were soaked in 80% ethanol for one week and then filtered using Whatman filter paper No. 42 to get the plant extract.

Experimental site

The animals were acclimatized in an animal house at the Biochemistry department at the Federal University of Owerri, under room temperature and relative humidity of 40-65% with a 12h natural light-dark cycle. The animals were granted free access to water and rats chew in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for animal experiments.

Experimental design

According to the Ezejiofor & Okoroafor [12], the median lethal dose (LD50) of the plant extract was established as 1600mg/kg, this led to the adoption of safe doses of 250mg/kg and 500mg/kg.

Thirty (30) male albino Wistar rats used for this experiment were grouped into five (5) groups, six (6) for each group;

Group NC, Negative control: Rats fed normal diet and water

Group PC, Positive control: Untreated rats fed with high-fat diet and water

Group LDE: High-fat diet fed rats treated with 250mg/kg body weight of ethanolic leaf extract of Diodia sarmentosa

Group HDE: High-fat diet fed rats treated with 500mg/kg body weight of ethanolic leaf extract of Diodia sarmentosa

Group SIM: High-fat diet fed rats treated with 5mg/kg body weight of Simvastatin. The efficacy of the extract was compared to Simvastatin (a statin-derived drug) used in reducing the risk of cardiovascular diseases.

Treatment and sample collection phase

The rats were placed on High-fat diet (Ghee and Coconut oil in the ratio of 3:1) for six (6) weeks adapted from a previous study by Munshi et al. [15] except for the negative control group. All animals were allowed access to food and water and the body weight of rats was taken weekly.

From the 4^th week till the end of the experiment, groups PC, LDE, HDE, and SIM continued with high-fat diet and their respective doses of extract and drug respectively, while group NC remained on normal rat chew. The appropriate dosages of the extract and drug were administered to the animals orally once daily by intubation using an intravenous cannula tube. The rats were allowed to fast for Twenty four hours, after the last treatment, they were anesthetized with chloroform vapour and sacrificed. The hearts from the sacrificed animals were removed and washed with ice-cold saline, blotted dry, and stored at -20^oC. Heart tissues were homogenized, and put inside labeled tubes for biochemical analyses.

Determination of Total Protein Concentration

Total protein concentration was determined using the Biuret method as described by Gornall et al. [16] as reported by Hassan et al. [17] was employed for the determination of protein concentration.

Determination of Lactate dehydrogenase (LDH) activity

Lactate dehydrogenase activity was determined according to the method of Wroblewski & La Due [18] as reported by Oloyede & Sunmonu [19].

Determination of Lipid peroxidation

Lipid peroxidation as expressed as malondialdehyde levels in the heart tissue was determined spectrophotometrically by assessing the concentration of thiobarbituric acid reactive substances (TBARS) described by Buege & Aust [20] as reported by Nair et al. [21].

Determination of antioxidant enzymes

The assay of the antioxidant enzyme Superoxide dismutase (SOD) activity was carried out according to the procedure of Das et al. [21]. Catalase (CAT) activity in the samples was assayed following the procedure of Aebi [23] as reported by Hassan et al. [24]. Glutathione-S-transferase (GST) in the samples was measured by the method of Habig et al. [25] as reported by Sasi Bhusana Rao et al. [26]. Glutathione peroxidase (GPx) in the samples was measured by the method of Rotruck et al. [27] as reported by Sajeeth et al. [28].

Statistical Analysis

Data were analyzed using appropriate software (Microsoft Excel 2013). Results were presented as mean ± Standard deviation of four determinations and statistically analyzed using a one-way analysis of variance on a statistical computer software program (SPSS 21). The degree of statistical difference was accepted as significant at p < 0.05.

The results of superoxide dismutase (SOD) activities in the heart tissue across the study groups ranging from 19.37 ± 3.43 — 29.71 ± 2.77 (IU/mg Protein) is as presented in Table 1. The PC group showing the lowest activity, while the LDE showing the highest activity.

Table 1: Result of Superoxide dismutase activity in the various study groups

	Groups	Heart Tissue
SOD (IU/mg Protein)x10^-6	NC	26.29±1.65^b
	PC	19.37± 3.43^a
	LDE	29.71± 2.77^b
	HDE SIM	26.70± 2.56^b 24.62±2.19^a