INTRODUCTION:

Medicinal plants play a vital role in fulfilling the health and medicinal needs of approximately 70% of the population in both developed and developing countries, where they are widely used for the treatment of various diseases and ailments¹. Plants synthesize a broad range of chemical compounds, which are classified based on their chemical structure, natural or biosynthetic origin, and functional groups into primary and secondary metabolites.

Primary metabolites are directly involved in growth and development. They are broadly distributed in nature and are present in virtually all organisms. These include chlorophyll, amino acids, nucleotides, and other essential compounds that play key roles in metabolic processes such as photosynthesis, respiration, and nutrient assimilation. Additionally, primary metabolites are widely utilized as industrial raw materials and food additives¹.

Secondary metabolites, in contrast, are produced during secondary metabolic processes and are not directly involved in plant growth or development. However, they often function as biocatalysts and serve as a foundational source for the pharmaceutical industry due to their extensive medicinal properties². The most significant groups of secondary metabolites include alkaloids, tannins, flavonoids, phlobatannins, saponins, and cardiac glycosides, each performing specific biological roles; for instance, saponins exhibit antifungal activity³.

Owing to their pharmacological potential, plant parts are frequently screened for the presence of phytochemicals. The discovery of important phytochemicals may lead to their isolation, purification, and characterization, ultimately forming the basis for new pharmaceutical products. The success of such investigations is highly dependent on the type of solvent used during the extraction process⁴. Hence, the choice of solvent plays a crucial role in the phytochemical screening of plant materials.

Punica granatum, commonly known as pomegranate, belongs to the family Punicaceae⁵. It is widely cultivated in India and other arid regions of Southeast Asia⁶, the East Indies, and tropical Africa. For centuries, different parts of the plant—including the bark, leaves, and fruit—have been used in traditional medicine⁷. The therapeutic potential of pomegranate is extensive, including applications in the treatment and prevention of cancers, cardiovascular disorders, diabetes, dental issues, and erectile dysfunction. The pericarp of P. granatum has traditionally been used to treat infections of the reproductive system, allergic dermatitis, tympanitis, scalds, diarrhoea, and dysentery⁸.

Considering the wide range of therapeutic applications associated with this plant, the present study was undertaken to investigate the presence of various phytochemicals in the leaf extract of Punica granatum, a plant known for its diverse medicinal properties⁹.

Nutritional Content:

Table 1: Nutritional Value of Pomegranate seeds

Sr. No.	Nutrition	Content
1	Energy	72Calories
2	Carbohydrates	16.3g
3	Fat	1g
4	Protein	1.5g
5	Fibres	3.5g
6	Sugar	11.9G
7	Vit-C	8.9mg (14.8%DV)
8	Vit-K	14.3mg (17.9%DV)
9	Foliate	33mg (8.3%DV)
10	Potssium	205mg (5.9%DV)
11	Phosphorus	31mg 3.1%DV)
12	Vit-B6	0.07mg (3.5%DV))

MATERIALS AND METHODS:

Preparation of Leaf Extracts:

Fresh leaves of the pomegranate plant (Punica granatum L.) were collected from Rahata, Maharashtra, India. The leaves were carefully separated, thoroughly washed with distilled water to remove dust and debris, and shade-dried. The dried leaves were then ground into a fine powder using a mechanical grinder.

Method of Extraction:

Decoction Method: A total of 50 grams of the dried leaf powder was accurately weighed and extracted with 500 ml of deionized or distilled water at 100°C for 40 minutes using a water bath. The extract was filtered through muslin cloth followed by Whatman No.1 filter paper. The filtrate was then evaporated to dryness under controlled temperature (30–40°C). The dried extract was stored in airtight containers in a cool, dry place for further analysis.

Phytochemical Screening:

The methanolic leaf extract of Punica granatum (PMLE) was subjected to preliminary phytochemical screening to identify the presence of various classes of phytochemical constituents using standard qualitative and quantitative methods.

Qualitative Phytochemical Screening Test for Anthraquinones:

To 1ml of the extract, a few drops of 1% hydrochloric acid were added. The formation of a red precipitate indicated the presence of anthraquinones.

Test for Carbohydrates:

To 1ml of the extract, α-naphthol solution was added. Concentrated sulfuric acid (H₂SO₄) was then carefully added along the sides of the test tube. The appearance of a violet ring at the interface confirmed the presence of carbohydrates.

Test for Reducing Sugars:

To 1ml of the extract, a few drops of Benedict’s reagent were added. The mixture was heated in a boiling water bath. The formation of a reddish-brown precipitate indicated the presence of reducing sugars.

Test for Flavonoids:

To 1ml of the extract, a few drops of 20% sodium hydroxide (NaOH) solution were added. The formation of an intense yellow color that became colorless upon the addition of dilute hydrochloric acid (HCl) indicated the presence of flavonoids.

Test for Phenols:

A portion of the extract was treated with 5% aqueous ferric chloride solution. The development of a deep blue or black color indicated the presence of phenolic compounds.

Test for Proteins:

To 1ml of the extract, 5ml of distilled water was added and the mixture was heated. Biuret reagent was then added. The appearance of a pink or violet color indicated the presence of proteins.

Test for Free Amino Acids:

The extract was heated with a 0.2% solution of ninhydrin. The formation of a purple color confirmed the presence of free amino acids.

Quantitative Phytochemical Screening:

Estimation of Total Phenolic Content (TPC):

The total phenolic content in the leaf extract was determined using the Folin-Ciocalteu method. A 0.2ml aliquot of the sample was mixed with 0.5ml of Folin-Ciocalteu reagent (diluted 1:2) and 4ml of 1M sodium carbonate solution. The mixture was allowed to stand for 30 minutes at room temperature. Absorbance was measured at 750nm using a UV-Visible spectrophotometer (Beckman DU 7400, USA). The total phenolic content was calculated and expressed as milligrams of chlorogenic acid equivalent per gram of dry weight of extract (mg CGE/g DW).

Estimation of Tannins:

Tannin content was estimated following the method of Peril and Pompeii (1971). A 0.5ml aliquot of the extract was diluted to 2ml with distilled water; an equal volume of water served as the blank. To this, 0.5ml of diluted Folin-Ciocalteu reagent (1:2) and 5ml of 35% sodium carbonate were added. The solution was allowed to stand at room temperature for 5 minutes, and the resulting blue coloration was measured at 640nm. Tannin content was calculated using a standard curve of chlorogenic acid and expressed as mg CGE/g DW.

Estimation of Total Flavonoid Content (TFC):

Total flavonoid content was determined using the aluminium chloride colorimetric assay. A 0.5ml aliquot of extract was mixed with 2ml of distilled water and 0.15 ml of 5% sodium nitrite (NaNO₂). After 6 minutes, 0.15 ml of 10% aluminium chloride (AlCl₃) was added, followed by incubation for another 6 minutes. Then, 2 ml of 4% sodium hydroxide (NaOH) was added, and the final volume was made up as necessary. The mixture was well mixed, and absorbance was measured at 510 nm. Total flavonoid content was calculated using a standard calibration curve of quercetin and expressed as mg quercetin equivalent per gram dry weight (mg QE/g DW).

Estimation of Total Triterpenoids:

To determine total triterpenoids, 200µl of the sample was taken in a 10ml volumetric flask and evaporated in a water bath. Then, 1 ml of 5% vanillin in acetic acid and 1.8ml of sulfuric acid were added. The mixture was incubated at 70°C for 30 minutes, cooled, and the volume was made up to 10ml with acetic acid. The absorbance was read at 573nm against a blank. The total triterpenoid content was calculated using ursolic acid as the standard and expressed as mg ursolic acid equivalent per gram dry weight (mg UE/g DW).

Estimation of Total Carbohydrates:

For carbohydrate estimation, 0.2ml of the extract was diluted to 1ml with distilled water. Then, 4 ml of anthrone reagent (0.2% anthrone in ice-cold concentrated sulfuric acid) was added. The mixture was heated in a boiling water bath for 8 minutes, cooled, and absorbance was recorded at 630nm. D-glucose was used as a standard, and the total carbohydrate content was expressed as a percentage of the dry weight.

Estimation of Protein Content:

A 0.2 ml aliquot of the extract was diluted to 1ml with distilled water. Then, 5ml of alkaline copper reagent was added and allowed to stand for 10 minutes. Subsequently, 0.5ml of Folin-Ciocalteu reagent was added, and the mixture was incubated in the dark for 30 minutes. Absorbance was measured at 660 nm. Bovine Serum Albumin (BSA) was used as the standard, and protein content was expressed as a percentage of dry weight.

Estimation of Total Lipids:

Ten grams of the dried sample were used for lipid extraction using 150ml of petroleum ether over 16 hours at a solvent condensation rate of 2–3 drops per second using a Soxhlet apparatus. The resulting extract was evaporated at room temperature to dryness. The weight of the dried extract was used to calculate the total lipid content, expressed as a percentage of the dry weight.

Estimation of Alkaloid Content (Harborne, 1973 Method):

Five grams of dried leaf powder were mixed with 200 ml of 10% acetic acid in ethanol in a 250 ml beaker, covered, and allowed to stand for 4 hours. The mixture was filtered, and the filtrate was concentrated to one-fourth of its original volume using a water bath. Concentrated ammonium hydroxide was added dropwise to the concentrated extract until precipitation was complete. The solution was allowed to settle, and the precipitate was collected by filtration, washed with dilute ammonium hydroxide, and dried. The weight of the dried residue (alkaloids) was expressed as a percentage of the dry weight.

RESULTS:

Qualitative Phytochemical Screening:

Table: 3 Qualitative Test

Phytochemicals Tested		Presence/Absence
Phenols		+
Flavonoids	Test(a):Flavones	+
Anthocyanin		+
Coumarin		+
Quinones		+
Anthraquionone		+
Steroids		+
Alkaloids	Dragendroff’sTest	+
	Mayer’sTest	+
(+) = PRESENCE (Test positive)(-)=ABSENCE(Test negative)

Quantitative Phytochemical Screening:

Total Phenolic Content (TPC):

The total phenolic content was found to be 84.26 ± 1.24 mg CGE/g DW. This high phenolic content suggests significant antioxidant potential of the leaf extract.

Tannin Content:

The total tannin content was estimated to be 32.45 ± 0.98 mg CGE/g DW, indicating the presence of potent polyphenolic compounds which may contribute to antimicrobial and anti-inflammatory activities.

Total Flavonoid Content (TFC):

The total flavonoid content was recorded as 46.87 ± 1.10 mg QE/g DW, revealing the plant’s potential role in free radical scavenging and other therapeutic effects.

Total Triterpenoids:

The total triterpenoid content in the extract was found to be 15.34 ± 0.76 mg UE/g DW, suggesting possible anti-inflammatory and anticancer properties.

Total Carbohydrate Content:

The carbohydrate content was estimated to be 18.62 ± 0.85% of the dry weight, reflecting the plant’s nutritional potential.

Protein Content:

The total protein content was calculated as 7.93 ± 0.47% of dry weight, indicating a moderate presence of bioavailable proteins.

Total Lipid Content:

The lipid content of the leaf extract was found to be 3.25 ± 0.22% of dry weight, consistent with the low-fat nature of plant-derived materials.

Alkaloid Content:

The total alkaloid content was determined to be 5.68 ± 0.31% of dry weight. Alkaloids are known for various pharmacological properties, including antimicrobial and analgesic effects.

CONCLUSION:

The present study provides comprehensive qualitative and quantitative data on the phytochemical profile of Punica granatum (Pomegranate) leaf extract, reinforcing its potential as a valuable source of bioactive compounds with significant therapeutic applications. The analysis revealed the presence of various phytochemicals, including phenols, flavonoids, alkaloids, anthraquinones, steroids, quinones, anthocyanins, and coumarins. These classes of secondary metabolites are widely recognized for their pharmacological properties, including antioxidant, anti-inflammatory, antimicrobial, and anticancer activities.

Quantitative phytochemical estimation further substantiated the medicinal relevance of P. granatum leaves. The total phenolic content (TPC) was recorded at 84.26 ± 1.24 mg CGE/g DW, indicating a rich presence of polyphenols, which are key contributors to antioxidant activity through their ability to neutralize free radicals and reduce oxidative stress. The tannin content was found to be 32.45 ± 0.98 mg CGE/g DW, supporting potential applications in antimicrobial and anti-inflammatory formulations, as tannins are known to form complexes with microbial proteins, thus inhibiting microbial growth.

Flavonoids, quantified at 46.87 ± 1.10 mg QE/g DW, are another class of antioxidants abundantly present in the extract. Their known therapeutic benefits include vasodilation, modulation of cell signaling pathways, and protective effects against cardiovascular diseases and cancers. The significant flavonoid content in the leaf extract suggests its potential in reducing oxidative damage and improving cellular function.

The total triterpenoid content, estimated at 15.34 ± 0.76 mg UE/g DW, further enhances the pharmacological potential of the extract. Triterpenoids are known for their anti-inflammatory, hepatoprotective, and anticancer activities. Their presence in P. granatum leaf extract supports its traditional use in treating inflammatory conditions and possibly even as a chemopreventive agent.

Moreover, the extract demonstrated appreciable nutritional value. The carbohydrate content was 18.62 ± 0.85%, while protein and lipid contents were 7.93 ± 0.47% and 3.25 ± 0.22%, respectively. These results suggest that P. granatum leaves can contribute to the nutritional profile of herbal formulations or dietary supplements. Although primarily evaluated for medicinal uses, such nutritional composition supports the dual role of the plant in nutrition and health.

Importantly, the presence of alkaloids (5.68 ± 0.31%) highlights the therapeutic diversity of the plant, as these compounds are known for their broad spectrum of pharmacological actions, including antimicrobial, analgesic, and anticancer effects. The combination of multiple bioactive phytochemicals may lead to synergistic interactions, enhancing the efficacy of herbal remedies based on P. granatum.

Overall, the study underscores the medicinal and nutritional importance of Punica granatum leaves, validating their traditional usage and paving the way for their incorporation in phytopharmaceutical products. Given the significant presence of phenolics, flavonoids, and other secondary metabolites, further studies, including bioassay-guided fractionation and pharmacological evaluation, are recommended to explore their specific mechanisms of action and therapeutic potential. The findings also justify the plant's inclusion in herbal pharmacopoeias and its possible exploitation in the development of natural health products and functional foods.

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Received on 15.04.2025 Revised on 10.05.2025

Accepted on 30.05.2025 Published on 25.07.2025

Available online from July 31, 2025

Res. J. Pharma. Dosage Forms and Tech.2025; 17(3):191-195.

DOI: 10.52711/0975-4377.2025.00027

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