Anti-Atherosclerotic Activity of Plumeria Acuminata Leaf Extract Against Triton X-100 Induced Hyperlipidemia Model in Rats

Introduction

The cardiovascular system (CVS) is responsible for transporting blood throughout the body through a well-organized, closed network of vessels. The heart, a muscular organ, serves as the central pump.1 One important factor in the development of cardiovascular diseases (CVDs) is oxidative stress, which occurs when there is a reduced supply of oxygenated blood to the cardiac muscle. The reactive oxygen species (ROS) can cause oxidative damage to cellular components, disrupting normal functioning and triggering disorders. Free radicals consume antioxidant enzymes, leading to lipid peroxidation and oxidation of lipoproteins, which play a role in atherosclerosis.2 Atherosclerosis is a long term disease that makes arteries thick and hard because of a buildup of plaque in the artery walls. Plaque is a sticky substance made of cholesterol, fat, calcium, blood cells, and other substances in the blood.³

Triton X-100, a non-ionic is extensively utilized to induce hyperlipidemia in animal models for the purpose of researching cholesterol and triacylglycerol metabolism. The accumulation of plasma lipids by this detergent is primarily attributed to the inhibition of lipoprotein lipase activity.⁴

Plumeria acuminata, is a member of the Apocynaceae family. This plant contains active phytochemical constit uents such as alkaloids, steroids, flavonoids, phenolics, tannins, and glycerides. Notably, lupeol acetate, ursolic acid, stigmast-7-enol and lupeol carboxylic acid have been isolated from its leaves.⁵ Of these, ursolic acid, a chemical constituent, exhibits anti-atherosclerotic activity.⁶

Since  the leaves of P.acuminata possess ursolic acid as a phytochemical constituent, along with antioxidant, free radical scavenging, and anti-inflammatory activities, it may possess anti-atherosclerosis effects. However, there is currently no literature available that confirms the anti-atherosclerosis activity of P.acuminata. Hence, the aim of the present study was to demonstrate anti-atherosclerotic activity of methanol extract of P.acuminata leaves in Triton X-100 induced hyperlipidemia model.

Materials and Methods

Drugs and chemicals

The fresh leaves of P.acuminata collected from local areas of Chikkaballapur, Karnataka, were authenticated by Central Ayurvedic Research Institute, Bengaluru with Code No: Authentication/SMPU/CARI/BNG/2023-24/2263. Tween 80 was procured from Avra Synthesis Pvt. Ltd. in Telangana. Atorvastatin was obtained as a complimentary sample from Biocon Ltd. in Bengaluru. Methanol and Triton X-100 were acquired from S D Finechem Limited, Mumbai. Furthermore, the Biochemical Parameter kits were sourced from Swemed Diagnostics.

Extract preparation

Before utilization, it was ensured that the leaves were free from microbial growth and contamination. Fresh leaves of the P.acuminata plant were meticulously collected, cleansed, and dried in the shade. Afterward, the substances were carefully transformed into a fine powder and stored in an airtight container until they were needed for further analysis. The powdered substance was then packed into the thimble of the Soxhlet, and subjected to extraction using 99% methanol as the solvent. The extraction process continued until the dark greenish colour of the solvent became colourless, after which solvent recovery was performed using a rotary evaporator.⁷

Animals

Albino Wistar rats of either sex weighing between 150 200 g were utilized for the study. All the animals were sourced from the Drug Testing Laboratory, Bengaluru. The rats were accommodated in polypropylene cages (32×16 cm) with stainless steel grill tops and were bedded with paddy husk and provided commercial pelleted rat food (Shri Venkateshwara Enterprises, Bangalore). The studies were conducted following the Committee for Control and Supervision of Experiments on Animals guidelines and received approval from the Institutional Animal Ethics Committee under reference no. GCP/IAEC/DOP/2023-2024/97, dated 07/10/2023.

Acute oral toxicity study

This study was conducted following the Organisation for Economic co-operation and development guideline 423, using female rats weighing 150 to 200 g maintained in standard laboratory conditions. The maximum upper limit dose 2000 mg/kg (b.w.) of P.acuminata leaf extract was orally administered to three female rats, and their behaviour and signs of discomfort and nervous manifestations were assessed and thereafter observation continued for 14 days to detect delayed toxicity.⁸

Experimental protocol for Triton X-100 induced hyperlipidemia model

Hyperlipidemia was induced in Wistar albino rats through the administration of a freshly prepared solu tion of Triton X-100 via a single intraperitoneal dose 100 mg/kg after an 18 hour overnight fasting.⁹ Animals were divided into seven groups with each group containing six animals. The animals in group 1 and 2 were administered with vehicle (5% tween 80) and Triton X-100, respectively. Animals in groups 3, 4, 5, 6 and 7 were pretreated with Atorvastatin (10 mg/kg), Ursolic acid (50 mg/kg), different doses of methanolic extract of P. acuminata leaves (MEPAL), respectively which was suspended in vehicle (5% Tween 80) for seven days. After fasting for 18 hours, Triton X-100 was administered via intraperitoneal route, except group 1. After a day from the last dose of Triton X-100 administration, the blood was drawn from all rats via retro-orbital sinus under mild anesthesia. The animals were then sacrificed with an excess of anesthesia, and liver was collected for histopathological studies to assess fat deposition in the liver. Serum was separated from blood using cooling centrifuge at 3000 rpm for 10 min at 4°C. Total Cholesterol (TC), Triglycerides (TG), High-densityl-ipoprotein Cholesterol (HDL Cholesterol),Aspartate Transaminase (AST), Alanine Transaminase (ALT),Alkaline Phosphatase (ALP),Lactate Dehydrogenase (LDH)and Creatine Phosphokinase (CPK)were estimated by fully automated auto analyser (Swemed Diagnostics, Bengaluru). Free fatty acids (FFA)was analyzed with a UV–Visible Spectrophotometer (SHIMADZU UV-1800).^10-18 LDL cholesterol and very low density lipo-protein cholesterol (VLDL Cholesterol) were calculated by standard meth od.¹⁹

Histopathological analysis of liver

Upon conclusion of the study, all rats were humanely euthanized with the use of approved anesthesia, and the livers were dissected out and immediately placed in a 10% buffered neutral formalin solution taken in a specimen storage container. After 24 hours of contact time, it was given for slide preparation and interpretation.²⁰

Statistical analysis

The data were analyzed using one-way ANOVA, followed by Tukey’s post-hoc test for multiple comparisons, employing software GraphPad Prism. Results were exhibited as Mean ± SEM. Statistical significance was indicated by *P <0.05, **P <0.01, ***P <0.001, ### P

The values are represented as Mean ± SEM, n=6. The statistical significance was determined using one-way ANOVA with Tukey’s multiple comparison employing software graph pad prism. All groups were compared to disease control. Here, * indicates significance at P<0.05, ** indicates high significance at P<0.01, *** indicates very high significance at P<0.001, and ns indicates nonsignificance.

Results

Preparation of methanolic extract of Plumeria acuminata leaves

The % yield of methanol extract of P. acuminata leaves was 16.30%, which displayed a semi-solid nature coupled with an intense green color.

Acute oral toxicity studies (LD50)

The acute toxicity evaluation confirmed no fatalities among the animals even at 2000 mg/kg dose, leading to conclusion that the extract is safe. According to the CCSEA guideline 423, 2000 mg/kg was considered the LD50. Consequently, 200 mg/kg, 100 mg/kg, and 400 mg/kg were selected as therapeutic, half the therapeutic and double the therapeutic dose for further studies.

Group 1: Vehicle control (A and B): Liver tissue showed normal central vein, hepatocytes, and  portal vein (50X) and normal central vein, hepatocytes and fatty vacuolations (droplets) (100X).

Group 2: Disease control (C, D, E and F): Hepatocytes showed

• Steatosis with biliary hyperplasia around portal vein -moderate: 3+ (50X)
• Micro vesicular steatosis with ballooning of hepatocytes as a degenerative change - Severe :4+ (100X)
• Ballooning of hepatocytes with extensive fatty vacuolations, biliary hyperplasia around portal vein - Severe :4+
• Ballooning of hepatocytes with biliary hyperplasia around portal vein and pericholangitis - moderate: 3+ (100X) with inflammatory cells micro vesicular.

Group 3: Atorvastatin group (G and H) : Liver tissue showed normal central vein, hepatocytes and portal vein (50X) and normal central vein, hepatocytes, portal vein and fatty vacuolation (100X)

Group 4: Ursolic acid group (I and J): Liver tissue showed normal central vein and hepatocytes (50X and 100X)

Group 5: Treatment group 100 mg/kg (K and L): Liver tissue showed normal hepatocytes with fatty vacuola tions: mild:2+(50X) as well as normal hepatocytes with inflammation, central vein (100X)

 Group 6: Treatment group 200 mg/kg (M and N): Liver tissue showed normal central vein, hepatocytes and portal vein (50X) as well as normal central vein, hepatocytes and portal vein (100X)

Group 7: Treatment group 400 mg/kg (O and P): Liver tissue showed normal central vein, hepatocytes and portal vein (X50) as well as normal central vein, hepatocytes (X100)

Discussion

Atherosclerosis is a vascular disease caused due to atherosclerotic plaque accumulation in the Tunica intima of vasculature. These atherosclerotic plaques are rich in fatty contents like lipids. Hyperlipidemia is the important factor for atherogenesis. As mentioned, atherosclerosis plaques are rich in lipids caused due to the enhanced circulating plasma lipids like Cholesterol, Triglycerides, LDL-Cholesterol, and VLDL Cholesterol.²⁰

Triton X-100 has been shown to hinder the removal of triglyceride-rich lipoproteins, leading to acute hyperlipidemia in various animal studies. The administration of Triton X-100 resulted in increased plasma levels of triglycerides and cholesterol, primarily due to enhanced secretion of very low-density lipoprotein (VLDL) from the liver and a reduced breakdown of both VLDL and low-density lipoprotein (LDL).²¹

Serum parameters like total Cholesterol, Triglycerides, LDL, and VLDL levels were significantly (P<0.001) increased, while the levels of HDL-C were decreased.²² Additionally, the levels of ALT, AST, ALP, LDH, and CPK were significantly (P <0.001) increased in the disease control group (Triton X-100) compared to the control group.23 Atorvastatin has shown a significant (P<0.001) decrease in Total Cholesterol, Triglycerides, LDL, VLDL, ALT, AST, ALP, LDH, and CPK, and increased levels of HDL-C compared to the group treated with Triton X-100. Ursolic acid has shown a significant (P <0.001) decrease in total cholesterol, triglycerides, LDL and VLDL levels, while also increasing HDL-C levels.24 Additionally, it decreased ALT and AST levels in relation to the Triton X-100 group.²⁵ Ursolic acid also demonstrated a significant (P<0.001) decrease in the parameters such as ALP, LDH, and CPK in relation to the Triton X-100 group. Pretreatment of MEPAL 100 mg/kg, 200 mg/kg and 400 mg/kg has shown significant decrease in the parameters like total Cholesterol, Triglycerides, LDL, VLDL, ALT, AST, ALP, LDH and CPK and increased levels of HDL-C in relation to the Triton X-100 group. FFA levels were significantly increased in disease control (Triton X-100) as compared to control group FFA levels. MEPAL 100 mg/kg, 200 mg/ kg and 400 mg/kg has shown significant decrease in FFA (P <0.05), (P <0.001), (P <0.001), respectively when compared to Triton X-100 treated group. Atorvastatin and Ursolic acid have shown significant decrease in the parameter FFA (P <0.001), (P <0.05), respectively when compared to disease control (Triton X-100) group. Treatment group 200 mg/kg and 400 mg/kg of MEPAL showed the most significant (P <0.001) improvement.

Conclusion

This study revealed that the methanol extract of Plumeria acuminata leaves (MEPAL) demonstrated anti-atherosclerotic activity in Albino Wistar rats. MEPAL, particularly at 400mg/kg, significantly enhanced serum parameters and normal histopathology. Further investigation is required to understand its mechanisms and clinical applications. This study contributes valuable knowledge toward developing natural remedies for atherosclerosis disease.

Conflict of interest

The authors states that there is absence of conflict of interest.

Acknowledgement

The authors thank the Principal of the Government College of Pharmacy for essential facilities to conduct the research