Pharmacological evaluation of Vernonia elaeagnifolia (Asteraceae) leaves in hyperlipidemic albino rabbits

Purpose: To evaluate the antihyperlipidemic efficacy and phytochemical constituents of Vernonia elaeagnifolia aqueous leaf extract. Method: Qualitative phytochemical analysis of V. elaeagnifolia leaves was performed. Thirty healthy albino rabbits were divided into six groups (n = 6). Cholesterol powder (0.5 g/kg) in 10 mL coconut oil (vehicle) was given orally to induce hyperlipidemia. The aqueous leaf extract of Vernonia elaeagnifolia was administered at 250 mg/kg and 500 mg/kg per oral. Lipid profile, hepatic enzymes and oxidative stress markers were evaluated. Results: Phytochemical screening indicated the presence of tannins, proteins, flavonoids, phenols, alkaloids and saponins. Oral administration of cholesterol powder significantly (p < 0.05) raised the level of serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c) and triglyceride (TG) along with significant (p < 0.05) decrease in serum concentration of high-density lipoprotein cholesterol (HDLc). Concentration of serum TC, LDL-c, TG and liver enzymes was significantly reduced in V. elaeagnifolia-treated groups. The levels of oxidative stress markers were restored to normal when the animals were treated with V. elaeagnifolia leaf extract; increased levels of antioxidant enzymes were observed. Conclusion: The aqueous leaf extract of V. elaeagnifolia possesses antihyperlipidemic and antioxidant potentials that are dose-dependent. However, further studies are required to develop the plant for therapeutic applications.


INTRODUCTION
Variations in normal functions of body alter the homeostasis which may lead towards serious health problems such as hypercholesteremia [1]. Hyperlipidemia is a heterogeneous metabolic disorder. It plays an important role in the progression of life threatening cardiovascular diseases such as myocardial infarction, cardiac arrest and angina. The major accounting problems of hyperlipidemia are increase in serum cholesterol level and oxidative stress [2]. Plaque formation takes place in the blood vessels when excess of LDL cholesterol gets accumulated in the endothelial spaces. It may worsen hypertension and normal functioning of liver and kidney [3,4].
Various types of antihyperlipidemic drugs are being used e.g. Statins (simvastatin, atorvastatin etc.), fibrates, ezetimibe, nicotinic acid and fish oils (omega-3 fatty acids) in order to decrease the serum cholesterol level [5]. Use of these synthetic medications may lead towards severe irregularities including abnormal liver function, hyperuricemia and dry skin [6]. Statins are involved in increasing hepatic transaminase level in the serum upon prolonged use [7].
Medicinal plants have been widely used in Ayurveda system of medicine. Evidences from literature favor the use of medicinal plants because they increase the immunity of body against various diseases [8]. V. elaeagnifolia (Asteraceae) is a creeper. It is mostly cultivated in Asia and Europe. Plants of this family have medicinal properties against upper respiratory tract infections, stomach ulceration and skin infections [9]. Chemical constituents found in V. elaeagnifolia ethanolic extract are flavonoids, phenolic compounds, tannins, terpenoids, phytosterols, alkaloids, and coumarins. Conventionally V. elaeagnifolia leaves are being used as leech repellent [10].
Current investigation was designed to investigate the anti-hyperlipidemic activity of aqueous extract of V. elaeagnifolia leaves.

EXPERIMENTAL
Animals 30 healthy albino rabbits (aged, 1 year; body weight, 2.0 ± 0.2 kg) of either sex were procured from local market of Faisalabad, Pakistan. The rabbits were kept in iron cages at temperature of 25º C and 12 h dark/12 h light cycle. The rabbits were on normal feed (lucern) and water ad libitum. Compliance with the rules was made by following Principles of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) [11]. All the trials conducted on animals were approved from the Committee of Animal Experimentation by the use of First Affiliated Brooke Hospital University of Agriculture (approval ref no. XW0642/2015).

Plant material
The leaves of V. elaeagnifolia were obtained from local market of Faisalabad, Pakistan. These were identified from Department of Botany, University of Agriculture, Faisalabad. In order to remove dust from leaves, they were washed with tap water. Afterwards leaves were dried in shade. Dried leaves were ground in electric grinder to have fine powder. The grounded material was stored at 25º C in air tight container.

Animal studies
Animals were categorized into 5 groups where each group was containing 6 animals. Group 1 was normal group in which animals were fed on routine diet only; Group 2 was hyperlipidemic control and received cholesterol (0.5 g/kg) in coconut oil (10 ml); Group 3 was positive control and received cholesterol powder + atorvastatin (0.25 mg/kg) p.o; Group 4 was treated group I and received cholesterol powder + V. elaeagnifolia aqueous extract (250 mg/kg) p.o; group 5 was treated group II and received cholesterol powder + V. elaeagnifolia aqueous extract (500 mg/kg) orally.

Proximate composition
Proximate composition (moisture content, dry matter, ether extract, total ash, crude protein and crude fiber) of V. elaeagnifolia leaves was determined according to various methods [13].

Elemental determination
One g of the powdered sample of V. elaeagnifolia leaves was digested with digestion mixture (nitric acid and perchloric acid) at 180 -200 ºC. Resulting transparent solvent was diluted with double distilled water up to 100 mL. This digested solution was used to determine elements through Atomic Absorption spectrophotometry (AAS; Hitachi Polarized Zeeman, Z-8200, Japan), according to the protocols described in AOAC (Association of Official Analytical Chemists; 1990).

Phytochemical screening
Phytochemical screening of V. elaeagnifolia leaves was done according to standard protocols for proteins, alkaloids, tannins, fixed oils, saponins and flavonoids [14].

Preparation of extract
Dried powder of leaves was added into soxhlet apparatus along with petroleum ether and extraction was carried out. The extract was subjected to hot water bath for evaporation.
Dried residue was collected and used as antihyperlipidemic drug [12].

Biochemical analysis
Blood samples were collected from jugular vein located in the neck via venous puncture. Blood was centrifuged at 1000 rpm for 10 min to isolate serum. Serum total cholesterol (TC), triglyceride (TG), HDL-c, LDL-c, ALP, ALT, AST, TAC, MDA, TOS, Catalase and SOD were measured using standard biochemical kits (DiaSys Diagnostic Systems, USA).

Histopathological analysis
Animals were sacrificed via decapitation on 45 th day of experiment. Liver tissues were collected and fixed in neutral formalin solution. These tissues were processed for light microscopy according to reference methods for embedding, sectioning, mounting and staining with hematoxylin & eosin. Light microscope (Olympus optical Co., Tokyo, Japan) with 40 X objective was used to examine the tissue samples.

Statistical analysis
Statistical analysis was done by using one way ANOVA tailed by Duncan's Multiple Range test to determine statistical differences at 5% level of significance. SPSS software version 3.16 was used and results were expressed as mean ± SE (standard error).

Proximate analysis
Results for the proximate composition are summarized in Table 1.

Phytochemical constituents
Qualitative phytochemical analysis of V. elaeagnifolia evidenced the presence of saponins, proteins, phenols, flavonoids, tannins, and alkaloids as represented in Table 3.

Biochemical profile
The amount of serum total cholesterol (TC), and triglyceride was significantly (p < 0.05) raised after the administration of cholesterol powder among all groups in comparison to the normal control. Serum concentration of triglyceride and total cholesterol in atorvastatin and V. elaeagnifolia treated groups showed significant reduction in dose dependent manner ( Figure 1).
Serum concentration of LDL cholesterol was increased while significantly (p < 0.05) decreased in cholesterol treated groups. When treated with V. elaeagnifolia then it restored the levels of LDL cholesterol and HDL cholesterol in comparison to normal control ( Figure 2). Amount of hepatic enzymes; alanine transaminase, alkaline phosphatase and aspartate transaminase was elevated significantly (p < 0.05) in cholesterol treated groups, when treated with V. elaeagnifolia then concentration of hepatic enzymes was decreased near to the normal as shown in Table  4.     Oxidative stress markers, total oxidant status (TOS) and malondialdehyde (MDA) levels were increased when treated with cholesterol while the amount in serum was restored when V. elaeagnifolia was given. Administration of cholesterol increased the oxidative stress and decreased the concentration of antioxidant enzymes (Catalase; SOD) and total antioxidant capacity. Treatment with V. elaeagnifolia increased the antioxidant capacity and amount of antioxidant enzymes as represented in Table 5.
As shown in Figure 3, liver tissue from control rabbit showed distinctive and clear boundaries of hepatic cells. While lobular structure of hepatocytes was disrupted in hepatic tissue obtained from hyperlipidemic rabbit. In addition, necrotic and binucleate cells were present. In atorvastatin treated animal, hepatocytes were resized to normal with lesser binucleate cells. When treated with 250 mg/kg of V. elaeagnifolia then it reversed the state of liver injury towards normal with few hazy hepatocytes. Appearance of hepatocytes in V. elaeagnifolia (500 mg/kg) treated rabbit was just alike to hepatic cells of atorvastatin treated animal. Parenchyma of cells appeared normal with mild degree of congestion ( Figure 3).

DISCUSSION
Current investigation was conducted to investigate the antihyperlipideic efficacy of V. elaeagnifolia aqueous leaf extract. For the induction of hyperlipidemia, cholesterol powder was administered to the animal models that increased the oxidative stress and serum cholesterol concentration. Hyperlipidemia is a major risk factor in the progression of cardiovascular diseases [15].
It was observed that micro and macro nutrients (calcium, manganese, magnesium, potassium, zinc, iron, cobalt and sodium) were present in V. elaeagnifolia leaves whereas heavy metals (lead, cadmium) were absent.
Previous research studies suggest that macro and micro nutrients have role in lowering the serum total cholesterol and free fatty acids. In another study it was observed that presence of calcium in sufficient amount in body enhances the process of fat digestion [16]. Calcium plays a major role in the formation of non-esterified linkages fatty acids in the small intestine. Calcium helps in the formation of insoluble soap with esterified fatty acids. This complex becomes more water soluble and readily gets eliminated from body via feces [17]. In one investigation it was found that micronutrients (zinc, manganese and copper) potentiate the antioxidant activity of super oxide dismutase, as SOD is involved in neutralizing lipid peroxides [18].
In the present study phytochemical screening indicated the presence of major phytoconstituents (flavonoids, proteins, alkaloids, tannins, saponins and tannins). It was observed in another research that presence of these phytoconstituents exert hypolipdemic effect. Previous literature claims that intake of saponin supplementation enhance the excretion of bile salts. Cholesterol is involved in the formation of bile salts. Thus excretion of bile salts results in decreased serum cholesterol. It was reported that flavonoids are phenolic compounds that potentiate the activity of lecithin acyl transferase (LCAT) enzyme, which in turn enhances the process of fat metabolism [19].
In present investigation, serum level of TC, LDL cholesterol and TG was elevated whereas HDL concentration was reduced when treated with cholesterol (0.5 g/kg). Serum concentration of TG, LDL cholesterol, TC and HDL-c was restored to normal level when treated with V. elaegnifolia aqueous leaf extract. These findings were supported by another research in which lemon juice was administered to hyperlipidemic animal model resulted in pronounced decrease in LDL cholesterol, TC and triglycerides [20].
V. elaeagnifolia significantly (p < 0.05) lowered the amount of TOS and MDA in the serum whereas level of antioxidant enzyme i.e. CAT and SOD was increased. Similar results were observed in another research when grape fruit juice was given to hyperlipidemic mice that resulted in decreased oxidative stress and increased serum level of antioxidant enzymes [21].
It was observed in the present study that V. elaeagnifolia reduced the levels of heptic enzymes i.e. aspartate transaminase, alkaline phosphatase and alanine transaminase. These findings were supported by previous research undertaken to evaluate antihyperlipidemic effect of ginseng extract. Administration of ginseng extract restored the serum levels of hepatic enzymes, in addition concentration of serum cholesterol was also reduced [22].

CONCLUSION
The findings of this study indicate that administration of V. elaeagnifolia aqueous leaf extract produced anti-hyperlipidemic and antioxidant effects in rabbits. However, further investigational studies are required to elucidate the cellular mechanisms involved in hypolipidemic activity of the extract.