Evaluation of Antidiabetic and Antihyperlipidemic Activity of Catharanthus pusillus on Alloxan-Induced Diabetic Albino Wistar Rats

Diabetes mellitus is a progressive metabolic disorder characterized by chronic hyperglycemia resulting from impaired insulin secretion, defective insulin action, or both. Extended hyperglycemia accelerates macrovascular and microvascular complications, frequently accompanied by secondary hyperlipidemia—a major driver of cardiovascular morbidity.

While conventional oral hypoglycemic agents (e.g., sulfonylureas, biguanides) and lipid-lowering drugs (e.g., statins) are widely used, their administration is often limited by adverse reactions, secondary failure, and cost constraints. Natural remedies offer a valuable source of alternative therapeutics. Catharanthus pusillus (Family: Apocynaceae) is a recognized medicinal plant with established antimicrobial and antioxidant properties, but its combined antidiabetic and antihyperlipidemic effects require formal preclinical evaluation.

2. MATERIALS AND METHODS

2.1 Plant Collection, Authentication, and Extraction

Fresh leaves of Catharanthus pusillus were collected from the region surrounding Jaipur, Rajasthan, India, and authenticated by a botanist. The leaves were shade-dried and coarsely pulverized. Powdered material (250 g ) was extracted with 70% ethanol in a Soxhlet apparatus at 60--70?C  for 35 hours. The filtrate was concentrated under reduced pressure at 40?C  to yield a dark green crystalline residue.

Percentage Yield (%)=Weight of Dried ExtractWeight of Raw Powdered Material×100

Percentage Yield=43.45 g250 g×100=17.38% w/w

2.2 Qualitative Phytochemical Screening

Standard qualitative biochemical assays were conducted to screen for primary and secondary plant metabolites.

2.3 Experimental Animals and Ethical Maintenance

Healthy Albino Wistar rats of either sex (200±50 g ) were housed under standard environmental conditions (25±2?C , relative humidity 50–60%, 12-hour light/dark cycle) with free access to standard pellet diet and water ad libitum, adhering to CPCSEA guidelines.

2.4 Induction of Diabetes and Secondary Hyperlipidemia

After an overnight fast, diabetes was induced via a single intraperitoneal injection of freshly prepared alloxan monohydrate (150 mg/kg  body weight in 0.9% normal saline). Animals were monitored for 24–48 hours for stable hyperglycemia. Rats exhibiting fasting blood glucose (FBG) levels >200 mg/dL  were included in the study. To establish hyperlipidemia, diabetic animals were concurrently maintained on a high-fat diet (HFD).

2.5 Experimental Animal Grouping

Animals were randomly assigned to five groups (n=6  per group):

• Group I (Normal Control): Healthy rats receiving vehicle (0.5 mL  distilled water/rat/day p.o.).
• Group II (Diabetic Control): Diabetic/HFD rats receiving vehicle (0.5 mL  distilled water/rat/day p.o.).
• Group III (Standard): Diabetic rats receiving Metformin (50 mg/kg  p.o.) + Atorvastatin (10 mg/kg  p.o.).
• Group IV (Extract Low Dose): Diabetic rats receiving C. pusillus extract (250 mg/kg  p.o.).
• Group V (Extract High Dose): Diabetic rats receiving C. pusillus extract (500 mg/kg  p.o.).

Daily oral treatment was administered for 28 consecutive days.

2.6 Biochemical Determinations

• Fasting Blood Glucose: Blood samples were collected from the retro-orbital plexus at Day 0, Day 7, Day 14, and Day 28. FBG was quantified using the Glucose Oxidase-Peroxidase (GOD-POD) enzymatic method.
• Lipid Profile Parameters: On Day 28, serum Total Cholesterol (TC), Triglycerides (TG), and High-Density Lipoprotein Cholesterol (HDL-C) were determined using commercial enzymatic assay kits (CHOD-PAP and GPO-Trinder methods) on a semi-automated biochemical analyzer. Low-Density Lipoprotein Cholesterol (LDL-C) was calculated using Friedewald’s formula:

LDL-C=Total Cholesterol-HDL-C-Triglycerides5

• Serum α -Amylase Activity: Measured at the conclusion of the 28-day treatment period using kinetic enzymatic methods.

2.7 Statistical Analysis

Data are expressed as Mean±Standard Error of the Mean (SEM)  (n=6 ). Statistical significance was evaluated using one-way Analysis of Variance (ANOVA) followed by Bonferroni’s post-hoc test. P<0.05  was considered statistically significant.

3. RESULTS

3.1 Extract Yield and Solubility Profile

The 70% ethanolic leaf extract yielded 17.38% w/w . The extract was soluble in water, methanol, and DMSO, partially soluble in chloroform, petroleum ether, and ethyl acetate, and insoluble in acetone.

3.2 Phytochemical Composition

Qualitative phytochemical screening confirmed the presence of alkaloids, carbohydrates, proteins/amino acids, flavonoids, cardiac glycosides, phenolics, tannins, and steroids.

3.3 Effect on Fasting Blood Glucose Levels

Alloxan administration induced severe hyperglycemia in Groups II–V (>390 mg/dL ). Daily oral administration of C. pusillus extract produced a time- and dose-dependent reduction in FBG over the 28-day period.

Values are Mean±SEM n=6. ?###?P<0.001 vs Normal Control; ?***?P<0.001 vs Diabetic Control.

By Day 28, the high-dose extract (500 mg/kg ) restored blood glucose levels (77.83±6.49 mg/dL ) to levels comparable to both the standard drug combination (74.33±4.76 mg/dL ) and normal control rats (78.17±3.31 mg/dL ).

3.4 Effect on Serum Lipid Profile

Diabetic control rats exhibited severe dyslipidemia, marked by significant elevations in TG, TC, and LDL-C alongside a reduction in HDL-C (P<0.001 ). Administration of C. pusillus extract for 28 days improved lipid parameters in a dose-dependent manner.

Values are Mean±SEM n=6. ?###?P<0.001 vs Normal Control; ?*P<0.05, ?**P<0.01, ?***?P<0.001, ns = not significant vs Diabetic Control.

3.5 Effect on Serum α -Amylase Activity

Alloxan-induced pancreatic islet injury led to a sharp drop in serum α -amylase levels in the diabetic control group (176.2±24.70 U/L ) compared to normal controls (589.0±16.45 U/L ; P<0.001 ). Treatment with C. pusillus extract for 28 days produced a dose-dependent recovery in serum α -amylase activity.