INTRODUCTION:

For tens of thousands of years, many plants have been used for their therapeutic qualities.

In India, plants have long been employed as herbal or natural remedies because they are abundant in pharmacologically active chemicals that are frequently used in at-home therapies for a variety of illnesses. Nearly 80% of the world's population receives their main healthcare from folk medicine, which includes many plant-based medical systems like Ayurveda, Siddha, Naturopathy, Tibetan medicine, and others. Many medicinal herbs, which are frequently used in conventional antidiabetic and antihyperlipidemic treatments, have been acknowledged internationally for their potential advantages in the management of diabetes. These plants' capacity to improve pancreatic function—either by boosting insulin secretion or by preventing the intestinal absorption of glucose—is the main source of their antihyperglycemic qualities. These plants may also support insulin-dependent metabolic activities.⁶

Despite the fact that there are more than 400 plant species with known hypoglycemic effects in the literature, the search for novel antidiabetic drugs derived from natural sources is still attractive because of their potential for safety and alternative therapeutic benefits in the treatment of diabetes. Numerous bioactive substances, such as glycosides, alkaloids, terpenoids, flavonoids, and carotenoids, are abundant in many of these plants and are commonly linked to their antidiabetic effects.¹⁵ For Hindus, Aegle marmelos, also known as bael or wood apple, is one such plant that has great religious significance. Since ancient times, its leaves have been offered to Shiva and Parvati in prayers. The three-pronged leaves of the plant represent the three Thrimurthies, Shiva, Vishnu, and Brahma. Each of them is holding leaflets that resemble the Thrisoolam, Lord Shiva's weapon. The three essential elements of creation, preservation, and destruction are linked to this particular leaf. Because of its varied phytochemical makeup, which includes alkaloids, tannins, essential oils, gums, resins, coumarin, and polysaccharides, Aegle marmelos is well-known for its efficacy in treating a range of medical conditions. Compared to many other fruits, it has a far higher nutritional content. Aegle marmelos leaves can be used in large amounts in conjunction with oral hypoglycemic medications to assist normalize blood glucose levels in those whose diabetes is not managed by these drugs or who have negative side effects from higher dosages.¹¹

Aegle marmelos is also very important in environmental settings. Ancient writings like the Rigveda, Yajurveda, Atharvaveda, Charak Samhita, and Sushrut Samhita have made mention of these herbs and emphasized their potential for healing a variety of ailments. The Rutaceae family includes the medium-sized Aegle marmelos tree, which grows to a height of 12 to 15 meters at a moderate rate. It has spreading branches that may occasionally have thorns and a small trunk with thick, soft, peeling bark. Alternate leaves on this deciduous tree can be clustered or single. Three to five oval, pointy leaflets, each approximately 10 cm long and 2 to 5cm wide, with shallow teeth around the margins, make up the leaves. Notably, a lengthy petiole connects the terminal leaflet.

A variety of metabolic diseases collectively referred to as diabetes mellitus are typified by persistently elevated blood sugar (hyperglycemia) brought on by issues with insulin secretion, action, or both. Anabolic hormone insulin has a critical role in controlling the metabolism of proteins, fats, and carbs.¹⁷

Metabolic imbalances arise when tissues (such as muscle and fat) become resistant to the effects of insulin or when insulin levels are inadequate.

The type and duration of diabetes can affect how severe the symptoms are.

In the early stages, many people, particularly those with type 2 diabetes, may not exhibit any symptoms. Nonetheless, certain people may have serious symptoms, especially kids with type 1 diabetes, who frequently exhibit symptoms like increased thirst, increased hunger, weight loss, blurred vision, and increased urination^.[16]

Serious consequences from diabetes, such as diabetic ketoacidosis or nonketotic hyperosmolar syndrome, can cause stupor, coma, or even death if untreated. Controlling blood sugar levels is crucial to avoiding these serious consequences.²⁰

A comprehensive strategy including medication and lifestyle changes is necessary for the effective management of type 2 diabetes mellitus (T2DM). The following are important factors to take into account in order to achieve and sustain appropriate metabolic control:

· Diet: It's crucial to have a balanced diet full of fruits, vegetables, whole grains, lean meats, and healthy fats. Blood glucose control can be achieved by keeping an eye on carbohydrate consumption.

· Physical Activity: Consistent exercise helps control weight and enhances insulin sensitivity. Every week, try to get in at least 150 minutes of moderate-intensity exercise.

· Weight management: Maintaining a healthy weight can lower the risk of problems and greatly enhance glycemic control.

· Drugs: Sulfonyl urea, metformin, etc.

· Encouraging patients to take an active role in their health requires educating them about their disease, available treatments, and self-management techniques.

· Counseling and support groups can also aid in motivation and medication and lifestyle adherence.

In addition to aiding in glycemic control, combining proper pharmaceutical therapy with lifestyle modifications is crucial in minimizing complications related to diabetes. Blood glucose levels must be regularly monitored in order to evaluate the efficacy of treatment and make any required modifications^.3

Fig.1: Aegle marmelos leaves

Classification:

· Domain: Eukaryote

· Kingdom: Plantae

· Subkingdom: Tracheobionta

· Division: Magnoloiphyta

· Class: Magnoliophyta

· Subclass: Rosidae

· Order: Sapindales

· Family: Rutaceae

· Genus: Aegle

· Species: Aegle marmelos

1. Hypoglycemic Activity:

· It has been demonstrated that bael leaves reduce blood glucose levels in both humans and animals with diabetes. There are several theories as to how this action works, including: Inducing pancreatic beta cells to secrete more insulin.¹⁰

· Improving tissue glucose uptake, which lowers blood glucose levels; Preventing intestinal glucose absorption, which lessens postprandial (after meal) blood sugar increases.

· When administered to diabetic pancreatic patients, leaf extract increased beta cell function and aided in the regeneration of streptozotocin-damaged pancreatic tissue. In certain patients with diabetes mellitus, administering leaf extract for 15 days resulted in a considerable reduction in blood cholesterol levels and a small drop in blood glucose levels in an uncontrolled clinical experiment^.1

2. Anti-Inflammatory Properties:

· Insulin resistance and the emergence of complications from diabetes are largely caused by chronic inflammation. Bioactive substances with anti-inflammatory qualities found in bael leaves aid in lowering inflammation in the body, which may enhance insulin function and avert problems.

· Both traditional knowledge and contemporary scientific study support the prospective anti-inflammatory properties of bael leaves. Bael leaves are a potentially useful natural treatment for inflammatory disorders because of their components, especially flavonoids and other polyphenolic chemicals, which may help control the inflammatory response. To completely verify their effectiveness and safety for human health, more research and clinical trials are necessary.¹⁴

3. Antimicrobial and Antifungal Properties:

· Because of its potent antibacterial and antifungal properties, Aegle marmelos is useful in treating bacterial, fungal, and viral illnesses. It has long been used to treat: Skin infections, where its antimicrobial qualities can aid in the treatment of wounds, abscesses, and fungal infections like ringworm; Gastrointestinal infections, including cholera, dysentery, and diarrhea, because it inhibits harmful bacteria like Shigella and E. coli. • By preventing microbial growth in the respiratory system, it helps prevent respiratory infections such colds, coughs, and throat infections.²

· Numerous animal and human fungi, including Trichophyton mentagrophytes, T. rubrum, Microsporum gypseum, M. audouinii, M. cookei, Epidermophyton floccosum, Aspergillus niger, A. flavus, and Histoplasma capsulatum, have been shown to be susceptible to the antifungal activity of the essential oil extracted from the leaves of the bael tree. The seed's unsaponifiable matter has demonstrated significant in vitro action against a number of fungus, including Aspergillus fumigatus, Trichophyton rubrum, T. terrestre, Epidermophyton floccosum, A. niger and A. flavus.¹⁸

4. Hepatoprotective (Liver-Protecting) Properties:

· Hepatoprotective qualities found in Aegle marmelos' leaves and fruit aid in preserving liver health and averting liver damage. According to studies, bael: Guards against damage to the liver brought on by toxins, including those from chemicals, alcohol, and drugs.

· Promotes liver regeneration and aids in detoxification by strengthening the liver's antioxidant defense mechanism. Although more study, particularly human clinical trials, is necessary to completely establish its usage as a therapeutic agent for liver health, it may prove advantageous in controlling and preventing liver illnesses. As always, before utilizing any herbal medicines, especially for liver-related concerns, it is best to speak with a healthcare provider.⁷

5. Antioxidant Activity:

· One of the things that leads to the development of diabetes and its consequences is the oxidative stress that free radicals create. Antioxidants such as flavonoids, tannins, vitamin C, and beta-carotene, which are abundant in bael, help reduce oxidative damage to pancreatic beta cells and neutralize free radicals. By shielding these beta cells from oxidative stress, bael helps maintain the beta cells' capacity to produce insulin over time.¹²

6. Antibacterial Activity:

· The most adaptable unicellular pathogens are bacteria, which often spread through food, water, air, and soil and infect humans and animals. Bael is one of the natural compounds that could be used to treat these kinds of illnesses.

· There have been reports of certain bael leaf, root, and fruit extracts being effective against a wide range of bacterial species.¹³

· Pseudomonas salanacearum, Xanthomonas vesicatoria, Escherichia coli, and Aeromonas sp. were all susceptible to the essential oil extracted from the leaves. Vibrio cholerae, Salmonella typhimurium, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus have all been demonstrated to be susceptible to the root's ethanolic extract.¹⁹

Habitat and distribution:

Native to India, bael is a plant that grows well from the Himalayas to West Bengal. Its growth is mostly seen in the foothills of several states, including as Madhya Pradesh, Uttarakhand, Jharkhand, Bihar, Chhattisgarh, and Uttar Pradesh. The exotic spectrum of bael includes nations like Bangladesh, Egypt, Malaysia, Myanmar, Pakistan, Sri Lanka, and Thailand in addition to India. The bael plant has a unique flowering and fruiting cycle in India. Usually, the blooming season is in April and May, right after new leaves appear. The fruit then ripens throughout the course of 10 to 11 months, ripening between March and June of the following year.⁹

Fig 2: Aegle marmelos

MATERIAL AND METHODS:

· Leaves of Aegle marmelos were gathered from a nearby garden.

· The gathered leaves were thoroughly cleaned with running water from the faucet and allowed to dry in the shade. The leaves were coarsely pulverized after drying.

· The powdered 40g plant leaves were kept for 48 hours in a closed glass container with 500mL of methnol.

· After filtering, the extract was dried out in a vacuum evaporator at lowered pressure.³

Thin Layer Chromatography:

For TLC analysis, 10mg of Aegle marmelos ethanolic leaf extract per milliliter was dissolved in ethanol solvent. Silica Gel-G was used as an adsorbent in the preparation of TLC plates.

A appropriate amount of distilled water was combined with 100g of silica gel-G to create a slurry. The slurry was promptly transferred to a spreader, and plates of the appropriate size were created by spreading the slurry onto glass plates. After an hour of air drying, the plates were dried at 110°C for two hours to set the coating. About 10 µml of extracts were progressively put onto the plate using a micropipette, and they were allowed to air dry. Solvents such as ethyl acetate, acetone, and ultimately chloroform were used in the process: The 9:1 methanol ratio displayed clear bands.

Long UV and iodine-sprayed plates showed three main bands. The chromatograms were photographed and examined in the presence of light. The following formula was used to determine the Rf value.

Distance travelled by solute

Rf Value = ----------------------------------------

Distance travelled by solvent

The qualitative phytochemical screening of plant extracts often provides the crucial information about the chemical ingredients for the pharmacological and pathological discovery of novel medications. An outstanding finding that points to the existence of several phytochemicals is obtained by TLC profiling of an ethanolic leaf extract of Aegle marmelos. various solvent systems yield various Rf values for different phytochemicals. This variance in the phytochemicals' Rf values offers a crucial hint as to their polarity and aids in choosing the right solvent system for column chromatography's separation of pure substances. Low polarity compounds have a high Rf value in a less polar solvent system, while high polarity compounds have a low Rf value.⁴

Pure compounds can be separated from plant extract using a mixture of solvents with varying polarities in varying ratios. Only by examining the Rf values of components in various solvent systems can the proper solvent system for a given plant extract be chosen. TLC profiling of the ethanolic leaf extract of Aegle marmelos revealed three main bands on lengthy UV and iodine-sprayed plates under the current conditions. Rf was determined by dividing the solute's travel distance by the solvent's travel distance.¹

Phytochemical Analysis:

1. Test for Carbohydrates: One milliliter of Molisch's reagent and a few drops of strong sulfuric acid were added to two milliliters of extract. Carbohydrates were present when a purple color formed.

2. Test for Tannins: Two milliliters of 5% ferric chloride were added to one milliliter of extract. The development of a greenish-black hue suggested the presence of tannins.

3. Test for Saponins: Two milliliters of distilled water were added to two milliliters of extract, and the mixture was agitated for fifteen minutes lengthwise in a graduated cylinder. Saponins were present when a 1 cm layer of foam formed.

4. Test for Flavonoids: A fraction of the extract's aqueous filtrate was mixed with 5 milliliters of diluted ammonia solution, and then concentrated sulfuric acid was added. The presence of flavonoids was shown by the yellow coloration.

5. Test for Alkaloids: Two milliliters of strong hydrochloric acid were added to two milliliters of extract. The Mayer's reagent was then added in a few drops. The presence of alkaloids was indicated by the green color.

6. Test betacyanin and anthocyanin: 1ml of 2N sodium hydroxide was added to 2 ml of extract, and the mixture was heated to 100ºC for 5 minutes. The development of a yellow hue suggested that betacyanin was present.

7. Test for Quinones: One milliliter of pure sulfuric acid was applied to one milliliter of extract. The presence of quinones was shown by the formation of red hue.

8. Test for Glycosides: Three milliliters of chloroform and a 10% ammonia solution were added to two milliliters of extract. The development of a pink hue suggested the presence of glycosides.

9. Test for cardiac glycoside: Two milliliters of glacial acetic acid and a few drops of 5% ferric chloride were added to 0.5 milliliters of extract. This was covered with one milliliter of sulfuric acid concentrate. The presence of cardiac glycosides was revealed by the creation of brown rings at the contact.

10.Test for Terpenoids: Concentrated sulfuric acid was carefully added to 0.5 ml of extract after 2 ml of chloroform had been added. The presence of terpenoids was revealed by the reddish-brown color development at the contact.

11. Test for Triterpenoids: One milliliter of the Libemann-Buchard Reagent (acetic anhydride with concentrated sulfuric acid) was added to 1.5 milliliters of extract. Triterpenoids were present when a blue-green hue formed.

12. Test for Phenols: Two milliliters of distilled water and a few drops of 10% ferric chloride were added to one milliliter of the extract. The development of a green hue suggested the existence of phenols.

13. Test for coumarins: One milliliter of 10% sodium hydroxide was added to one milliliter of extract. The presence of coumarins was detected by the formation of a yellow tint.

14. Test for Acids: A sodium bicarbonate solution was applied to 1 milliliter of the extract. Effervescence was a sign that acids were present.⁸

Preformulation Studies:

1. Determination of Moisture Content

· Method: Weigh a 2 g sample in a pre-heated glass petriplate. Dry in an oven at 130 °C for 2 hours. Weigh before and after drying to calculate moisture content.

· Formula:

Moisture Content (%) = (W1−W2)/weight of sample×100

Where:

W1 = initial weight

W2 = weight after drying

2. Determination of Ash Content:

Method: Weigh the empty crucible. Weigh approximately 2g into crucible.

Weigh the crucible alog with powder. heat the sample in dessicator at high temp for 1hr.Cool in desiccator and weigh within 1h after reaching room temperature. Weigh ashed sample and record weight Calculate % Ash and record the value.

Formula:

Ash (%) = Weight of Ash/weight of sample×100

3. Angle of Repose:

The fixed funnel method is used to determine the angle of repose. Poured the powder sample through the funnel, continue the pouring until the pile reaches to consistent height approximately 2-3cm. Stop pouring and gently tap the funnel to remove any excess powder. Measure the height (h) of the pile and radius(r) of the base. Calculate the angle of repose using the formula.

Formula:

tan(θ)=h/r

Flow property	Angle of repose
25-30	Excellent
31-35	Good
36-40	Fair
41-45	Passable
46-55	Poor
56-65	Very poor

4. Bulk Density:

· Method: Fill a 100mL container with 15g of powder without compacting. Measure the visible volume (V₀).

· Formula:

Bulk density = weight of powder/Bulk volume

5. Tapped Density:

· Method: A Known mass of powder is poured in a measuring cylinder. The cylinder is tapped on a hard surface for 100 times. Tap until the volume stabilized.

· Formula:

Tapped density=weight of powder/tapped volume

6. Carr’s Index (Compressibility Index):

· Method: Calculate using bulk and tapped densities.

Compressibility Index (%) = (Tapped Density-Bulk density) / (Tapped density) ×100

Flow property	Carr’s Index
0-10%	Excellent
11-15%	Good
16-20%	Fair
>20%	Poor

7. Hausner’s Ratio

· Method: Calculate using tapped density and bulk density.

Hausners ratio = (Tapped density) / (bulk density)

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Received on 16.01.2025 Revised on 25.02.2025

Accepted on 02.04.2025 Published on 15.05.2025

Available online from May 17, 2025

Research J. Science and Tech. 2025; 17(2):148-154.

DOI: 10.52711/2349-2988.2025.00021

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