Phytochemical Profiling and Antidiabetic Efficacy of Naregamia alata (Wight & Arn) Methanolic leaf extract in Streptozotocin-induced Diabetic Rats

Plants have long been a vital source of therapeutic agents in traditional systems of medicine, particularly in India. According to Bassam et al. (2012) ^[1], plant-derived compounds constitute a substantial portion of active ingredients in modern pharmaceuticals. Despite the growing use of synthetic drugs, many conventional medications—including laxatives, antibiotics and antimalarials—have plant-based origins. India, with its vast biodiversity, is home to nearly 12,000 medicinal plant species out of the approximately 248,000 known globally ^[2]. However, only about 10% of these have been scientifically explored for their phytochemical and pharmacological properties ^[3], underscoring the significant potential for future research.

Phytochemicals, are biologically active secondary metabolites found in various plant parts which serve as natural defence mechanisms in plants. Compounds such as alkaloids, flavonoids, terpenoids, phenols and saponins have shown significant pharmacological activities, including antioxidant, antimicrobial, anti-inflammatory and anticancer effects ^[4,5]. One major health concern is type 2 diabetes mellitus, whose global prevalence is rapidly increasing, especially in developing countries. According to the World Health Organization ^[6,7], over 366 million people are affected worldwide, with India reporting 31.7 million cases as early as 2000 ^[8]. Plant-derived inhibitors are increasingly recognized as effective and safer alternatives to synthetic drugs ^[9].

Naregamia alata Wight & Arn, a small shrub from the Meliaceae family endemic to peninsular India—particularly Kerala—holds significant ethnobotanical value. Traditionally used to treat rheumatism, asthma, ulcers, anaemia and skin disorders ^[10], it stands out for its medicinal potential within a largely woody plant family. Reported antioxidant, antimicrobial, anti-inflammatory and antidiabetic activities suggest its relevance in managing diabetic wounds, which involve oxidative stress, infection and chronic inflammation. This study aims to scientifically validate the phytochemical composition and antidiabetic potential of Naregamia alata in streptozotocin-induced diabetic rats, supporting its development as a standardized plant-based therapeutic.

MATERIALS AND METHODS

Collection and processing of Naregamia alata leaves

Naregamia alata leaves were collected from the Calicut University campus, Malappuram, Kerala, India. The leaves were pre-washed in deionised water and kept under shade until completely dried for milling.

Soxhlet extraction of Naregamia alata leaves

For the study, infusion method of Soxhlet Extraction had been adopted. The powdered herbs of Naregamia alata leaves were filled in the thimble and placed in the Soxhlet extractor. The extractor was filled with solvent solutions (methanol, acetone, petroleum ether and aqueous solvents) and the temperature of 60?C was set and left for 6hr. Slowly and steadily the temperature was increased up to 100?C. The extract from the thimble was collected in the round bottom flask kept in the heating mantle below by passing through a side arm tube ^[11,12].

Phytochemical compound identification of Naregamia alata leaf extracts using Thin Layer Chromatographic technique

The extracts of Naregamia alata were analysed using Thin-Layer Chromatography (TLC) to profile phytochemicals present. Samples were applied ½ inch from the base of activated TLC plates using a capillary tube. Plates were developed in a chamber containing a suitable solvent system until the mobile phase reached the top. After development, the solvent front was marked, the plates dried and phytochemical bands were visualized using specific spraying reagents corresponding to each compound class. This method follows current best practices in phytochemical profiling of plant extracts ^[13]. The spots observed on the chromatography plate were marked and the retention factor (Rf) value of each compound was calculated using the formula:

Rf = Distance travelled by the solvent (cm)

        Distance travelled by the sample (cm)

The details of the identified phytoconstituents, the mobile phase used and their corresponding spraying reagents are summarized in Table-1.1.

Table-1: Thin layer chromatography analysis of Naregamia alata leaf extract

Animals

Healthy adult male albino Wistar rats (150–200?g), in-house bred, were used for the study. Animals were housed in polypropylene cages with husk bedding under standard conditions (25?±?2?°C, 55?±?10% humidity and a 12:12 hr light–dark cycle) and provided with a standard pellet diet (Amrut rat and mice feed, Sangli, India) and free access to water. All experimental procedures were approved by the Institutional Animal Ethical Committee (IAEC) and conducted in accordance with current guidelines for the care and use of laboratory animals ^[14].

Acute toxicity studies

The acute toxicity of Naregamia alata methanolic leaf extract was evaluated using the fixed-dose procedure in accordance with OECD Guideline No. 420. Thirty fasted male albino mice (25–30?g, 10 weeks old) were randomly assigned into three groups (n=10 per group). Group I served as the control and received distilled water, while Group II and Group III received Naregamia alata methanolic leaf extract at doses of 250?mg/kg and 500?mg/kg body weight, respectively, along with regular feed. Animals were closely monitored at 2-, 6-, 24- and 48-hours post-administration for signs of toxicity or behavioural changes. Mortality was recorded for 24 hours. Body weight and hematological parameters were statistically compared between control and treated groups. Based on these findings, a safe dosage for further experimentation was determined ^[15].

Effect of methanol extract of Naregamia alata leaf on normoglycemic rats

Male rats (n = 3 per group) were assigned to: Group I (distilled water), Group II (glibenclamide 0.25?mg/kg?b.w.) and Group III (Naregamia alata methanol leaf extract 500?mg/kg orally). Blood glucose was measured at 0, 1, 2, 3 and 4?hr post?dosing via retro-orbital sampling under mild ether anaesthesia ^[16].

Effect of methanol extract of Naregamia alata leaf on oral glucose tolerance test in STZ-induced diabetic rats (OGTT)

Diabetic rats were allocated to four groups (n = 3): Group I (normal control–distilled water), Group II (diabetic control–distilled water), Group III (glibenclamide 0.25?mg/kg) and Group IV (Naregamia alata leaf extract 500?mg/kg). Thirty minutes post-treatment, all rats received glucose (2?g/kg). Blood samples were collected at 0, 30, 60, 90 and 120?min to assess post?prandial glucose response ^[17].

Evaluation of antidiabetic activity -Induction of diabetes

Diabetes was induced in rats by single intra peritoneal (i.p.) injection of Streptozotocin (STZ, Sigma chemical Co. USA) at a dose 60mg/kg b.w. freshly dissolved in 0.1M cold citrate buffer of pH 4.5; 48hr later blood samples were collected and blood glucose levels were determined to confirm the development of diabetes. Those animals which showed hyperglycemia (blood glucose levels >240mg/dl) were used in experiment ^[18].

Chronic treatment model

The rats were divided into 4 groups of 3 animals (n=3) each as below: Group I- Normal control (received distilled water 10ml/kg), Group II- Diabetic control untreated (received distilled water 10ml/kg), Group III-Diabetic treated with standard drug glibenclamide (0.25mg/kg/day), Group IV-Diabetic treated with Naregamia alata (500 mg/kg/day). For 30 days blood glucose levels and body weights were measured on 1^st, 10^th, 20^th and 30^th day of the study. Finally on day-30, blood was collected to estimate various parameters ^[19].

Estimation of plasma glucose, body weight and lipid profile

Every week, following overnight fasting (16hr fasting with free access to water), the blood samples were withdrawn from the animals by retro-orbital puncture under light ether anaesthesia. The plasma glucose estimation was done by the glucose oxidase/peroxidase (GOD/POD) ^[20] method using a standard kit obtained from Span Diagnostics, India. Body weight of all experimental animals was recorded using a digital weighing scale. The Triglycerides (TG), Total cholesterol (TC) and HDL levels ^[21] were estimated using standard kits obtained from Span Diagnostics, India ^[22].

VLDL = TG/5

LDL = TC − (HDL + VLDL)

Estimation of serum insulin

Serum insulin was measured via radioimmunoassay (RIA) using ¹²?I-labeled human insulin antibody (BRIT, BARC, India), which cross-reacts with rat insulin ^[23].

Estimation of liver glycogen

After 30 days of experimental period, the 12hr fasted rats were sacrificed by cervical decapitation. The liver tissue (1g) was collected, placed in a centrifuge tube containing 2ml of KOH (300g/L) after washing with saline water and heated for 20 min with occasional shaking. To this, a saturated solution of sodium sulphate (0.2ml) was added and mixed thoroughly. The glycogen was precipitated by the addition of ethanol (5ml). The precipitate was removed and dissolved in 10ml of water. 1ml of this solution was added to 1ml of HCl (1.2mol/l) and boiled for 2hr. After 2hr, the solutions were neutralized by NaOH (0.5mol/l) using phenol red as indicator. The neutralized solution was diluted to 5ml and transferred to a colorimeter tube and read at 620nm after adjusting the colorimeter with the reagent blank. The glycogen content was expressed as mg/g of liver tissue ^[24].

RESULTS AND DISCUSSION

Extraction of Naregamia alata leaf extracts

Naregamia alata leaf extracts were prepared using 4 different solvents (methanol, acetone, petroleum ether and aqueous solvents) in a standard Soxhlet apparatus. The extracts were found clear in screw cap bottles; stored in dark cool place to avoid sunlight and humid.

Investigation of phytochemicals and bioactive compounds using Thin Layer Chromatography

Following purification of Naregamia alata leaf extracts using silica gel column chromatography, the collected fractions were subjected to Thin-Layer Chromatography (TLC) to identify bioactive constituents. In the acetone extract, phenolic and terpenoid compounds were detected with Rf values of 0.67 and 0.69, respectively (Table-2, Fig-1). The methanol extract fractions revealed the presence of phenols (Rf?=?0.64), terpenoids (Rf?=?0.65) and flavonoids (Rf?=?0.67), as indicated by three distinct bands (Table-3, Fig-2). In the petroleum ether extract, phenolic and terpenoid compounds were identified with Rf values of 0.66 and 0.68, respectively (Table-4, Fig-3). The aqueous extract exhibited only phenolic compounds with an Rf value of 0.65 (Table-5, Fig-4). These results suggest the differential solubility and distribution of phytochemicals in various solvent systems, as reflected by their TLC profiles.

Table-2: Thin layer chromatography analysis of Naregamia alata leaf (Acetone extract)

+ Present, ++ Actively present, - Absent

Two spots identified for Phenol and Terpenoid

NO spots identified for Alkaloid and Flavonoid

Fig-1: Thin layer chromatography analysis of Naregamia alata leaf (Acetone extract)

Table-3: Thin layer chromatography analysis of Naregamia alata leaf (Methanol extract)

+ Present, ++ Actively present, - Absent

Three spots identified for Phenol, Terpenoid and Flavonoid

NO spots identified for Alkaloid

Fig- 2: Thin layer chromatography analysis of Naregamia alata (Methanol extract)

Table-4: Thin layer chromatography analysis of Naregamia alata leaf (Petroleum ether extract)

+ Present, ++ Actively present, - Absent

Two faint spots identified for Phenol and Terpenoid

NO spots identified for Alkaloid and Flavonoid

Fig- 3: Thin layer chromatography analysis of Naregamia alata leaf (Petroleum ether extract)

Table-5: Thin layer chromatography analysis of Naregamia alata leaf (Aqueous extract)

+ Present, ++ Actively present, - Absent

One faint spot identified for Phenol

NO spots identified for Alkaloid, Terpenoid and Flavonoid

Fig-4: Thin layer chromatography analysis of Naregamia alata leaf (Aqueous extract)

Acute toxicity Study

It was observed that Naregamia alata methanolic leaf extract was safe to use in animals and showed no mortality on 500 mg/kg b.w. Therefore 500 mg/kg dose was considered as a safe dose and selected for all in vivo experiments as maximal dose.

Effect of Naregamia alata methanol leaf extract in normoglycemic rats

The results from the study clearly indicated that there was no significant effect observed on normoglycemic rats when treated with the single dose of Naregamia alata methanol leaf extract (Table- 6).

Table – 6: Effect of Naregamia alata methanol leaf extract in normoglycemic rats

s – Statistically significant (p<0.05) when compared with normal

The results from the study clearly indicated that the methanolic extract of Naregamia alata leaves 500 mg/kg reduced the blood glucose level (hyperglycemia due to glucose load 2 g/kg) significantly and glibenclamide (0.25 mg/kg) after 60 min of oral administration, when compared to diabetic control (Table- 7).

Table – 7: Effect of Naregamia alata methanol leaf extract on oral glucose tolerance test in STZ-induced diabetic rats (OGTT)

s – Statistically significant (p<0.05) when compared with Group – II (diabetic control)

Hypoglycemic effect of the methanolic leaf extract of Naregamia alata

The results from the study clearly indicated that the methanolic leaf extract of Naregamia alata exhibited significant hypoglycemic activity in STZ-induced diabetic rats, whilst there was no significant effect observed on normoglycemic rats. However, at the end of 30 days of treatment, there was a 78.96% and 71.23% decrease of serum glucose levels with the glibenclamide and methanolic leaf extract (500 mg/kg) of Naregamia alata respectively when compared with diabetic control (42.13%) after 30 days (Table 8).

Table – 8: Effect of Naregamia alata leaf extract on serum glucose level

s – Statistically significant (p<0.05) when compared with Group – II (diabetic control)

Changes in body weight

At the end of 30 days treatment, the body weight of normal rats, Naregamia alata methanolic leaf extract and standard drug treated (Glibenclamide) group increased significantly; whereas body weight of diabetic control group decreased (Table- 9).

Table – 9: Effect of Naregamia alata leaf extract on body weight in STZ-induced diabetic rats

s – Statistically significant (p<0.05) when compared with Group – II (diabetic control) after 30 days

Lipid profile of animals treated with Naregamia alata methanolic leaf extract showed significant reductions of 20.24% CHL (Cholesterol), 89.84% LDL (Low Density Lipoproteins), 15.15% VLDL (Very Low-Density Lipoproteins), 39.66% TG (Triglycerides) after the treatment with methanolic leaf extract of Naregamia alata, respectively when compared with diabetic control rats. Also, there was a significant increase of HDL (29.75%) in the treated diabetic rats (Table- 10).

Table – 10: Effect of Naregamia alata leaf extract on serum lipid profile after 30 days

s – Statistically significant (p<0.05) when compared with Group – II (diabetic control)

Estimation of serum insulin and liver glycogen after 30 days

After 30 days treatment period it was observed that animals treated with Naregamia alata methanolic leaf extract showed a significant increase in the serum insulin level and liver glycogen level (Table- 11).

Table – 11: Effect of Naregamia alata extract on serum parameters after 30 days

s – Statistically significant (p<0.05) when compared with Group – II (diabetic control)