Agro-Waste Valorization of Pomegranate Peel: Isolation of Ellagic Acid and Development of an Antioxidant Cosmeceutical Cream

Natural products are essential in the process of finding and developing novel drugs. The peel of pomegranate (Punica granatum) is an important agricultural waste that contains a high amount of bioactive constituents, including flavonoids, tannins, and phenolic acids. Among these compounds, ellagic acid is notable for its potent radical neutralizing, Immunomodulatory, and antitumor properties. Therefore, the extraction and recovery of such valuable compounds using simple pharmaceutical methods like solvent extraction and chromatographic techniques are highly significant for both research purposes and industrial applications.

Ellagic acid is an endogenous polyphenolic substance, a member of hydrolysable tannins and is formed by the dimerization of gallic acid units. It is found in a variety of plant sources including pomegranate peel, berries, grapes, walnuts, and several medicinal plants. In plants, ellagic acid commonly exists in bound form as ellagitannins, which yield free ellagic acid upon hydrolysis.

MATERIALS AND METHODS

I. Sampling and Preparation of Sample

Pomegranates were purchased from a local fruit store. The peel was stripped off the fruit and used as the test material. The samples obtained were then rinsed using running tap water in order to remove the impurities, while the peels were dried in the sun for four days. After drying, the samples were milled into powder form using a miller machine and subsequently sieved with a No. 60 mesh sieve before storage in an airtight container.

II. Chemicals and Reagents

 pomegranate peel powder, beeswax, almond oil, borax, olive oil, sandalwood, distilled water, ethanol, Molisch’s reagent, concentrated sulfuric acid (H₂SO₄), Fehling’s solution A & B, Wagner’s reagent, lead acetate, ninhydrin reagent, diluted sulfuric acid, ferric chloride (FeCl₂), DPPH reagent, gallic acid, E. coli bacteria, rose water, 20% sodium carbonate solution, and ruthenium red dye.

III. Equipment and Apparatus

III. Extraction of compounds from pomegranate

Extraction Procedure of Ellagic Acid

Fresh peels of pomegranate were washed, dried under shade for 5 to 7 days and then ground into a fine powder form.   Approximately 50 to 100 grams of the powder obtained was defatted by means of petroleum ether through Soxhlet apparatus for a period of 4 to 6 hours.   The plant material was subjected to primary extraction with 70 to 100 percent methanol or ethanol through Soxhlet process for 6 to 8 hours till the solvent became clear. The extract obtained was concentrated with the help of rotary evaporator at a temperature ranging between 40 to 50 degrees centigrade.

TLC Method-

Thin Layer Chromatography (TLC) was performed for the identification of phytochemical constituents present in the extract.

• Stationary phase: Silica Gel G
• Mobile phase: Ethyl acetate: Formic acid: Acetic acid: Water (100:11:11:27)

Result

• Ellagic acid Rf ≈ 0.3–0.6

Column Chromatography

Materials Required

• Glass column (with stopcock)
• Silica gel (60–120 mesh)
• Solvents :
  • Petroleum ether / Toluene
  • Ethyl acetate
  • Methanol
• Sample: Pomegranate peel extract
• Cotton, sand, beaker, funnel
• TLC plates (for monitoring)

Procedure

Cotton wool and stationary phase are placed at the base of the column as a support for the stationary phase. Slurry of silica gel made in petroleum ether is slowly packed into the column, tapped to expel air bubbles, and topped with stationary phase. The extract is mixed with silica gel, made into dry flowing powder and applied on the column, then topped with sand. Fractionation is performed in an ascending manner by using eluents of increasing polarity starting with petroleum ether and toluene to ethyl acetate and methanol solutions, with a fixed flow rate. Eluates are collected in appropriately labeled test tubes of 10-20 mL each. The fractions are checked, and the ones that contain the ellagic acid compound can be distinguished based on their Rf values.

UV visible spectroscopy

The pomegranate peel extract and standard ellagic acid solution were prepared in methanol. The solutions were scanned in the UV-visible region between 200 and 800 nm using a UV-Visible spectrophotometer. The absorption spectrum of the extract was compared with the spectrum of standard ellagic acid. The presence of similar absorption peaks indicated the presence of ellagic acid and related phytoconstituents in the extract.

3. Sample Calculation (for 20 µg/mL)

0.8000.180​×100=22.50%

IC₅₀ Determination

• Plot Concentration vs % Inhibition

• From the graph, the concentration at which 50% inhibition occurs.

• Approximate IC₅₀ (from table):
• 60 µg/mL → 47.5%
• 80 µg/mL → 62.5%

    So IC₅₀ ≈ ~65–70 µg/mL

Estimation of total phenolic compounds present.

The reaction between phenols present in the extract and ferric ions (Fe³⁺), along with potassium ferricyanide, results in the formation of the Prussian blue complex. Initially, the extract is mixed with potassium ferricyanide, and ferric chloride is added in an acidic medium to generate the blue color. The intensity of this blue color varies depending on the concentration of phenolic compounds. After leaving the reaction mixture to rest for 15 to 30 minutes, the absorbance is determined at approximately 700 nm through the UV-Vis spectroscopy.

Antibacterial Bacterial Activity

To determine the anti-bacterial efficacy of the pomegranate peel extract, the Kirby-Bauer agar well diffusion test was used. The bacterium culture was inoculated into the nutrient agar plate. Bore holes of about 8 mm and then fill them with the extracts. Incubate for 24 hours at 37°C, after which time the anti-bacterial activity is assessed based on the size of the zone of inhibition produced.

Formulation of Anti-aging Cream-

Sr. No.	Ingredient	Role in Formulation
1	Beeswax	It functions as a thickening agent and emulsifier, aiding in the formation of a stable cream while also creating a protective layer on the skin.
2	Borax	Serves as an emulsifying agent; helps in blending oil and water phases and stabilizes the cream.
3	Almond Oil	Functions as an emollient; moisturizes and nourishes the skin, improves skin tone, and reduces dryness.
4	Olive Oil	Provides deep hydration; rich in antioxidants and helps in preventing skin aging and improving elasticity.
5	Sandalwood	Acts as a soothing and cooling agent; has anti-inflammatory and mild antimicrobial properties, and enhances skin texture and fragrance.

Formulation Composition of Anti-Aging Cream (B1–B3)

Sr. No.	Ingredients	B1	B2	B3
1	White Beeswax	4 g	4.3 g	4.5 g
2	Almond Oil	10 g	10 g	9.9g
3	Borax	0.14 g	0.17g	0.17 g
4	Punica Extract	1.5mL	1.5mL	1.5mL
5	Distilled Water	4.3 ml	3.8ml	4ml
6	Olive Oil	0.2ml	0.2ml	0.2ml
7	Sandalwood Powder	0.1 g	0.1 g	0.1 g
8	Rose Water	0.1 mL	0.1 mL	0.1 mL

Procedure for Preparation of Anti-Aging Cream

Weigh the required amounts of beeswax, almond oil, and olive oil, and heat them to melt the beeswax by putting them into a clean beaker through a water bath. Prepare the aqueous phase by dissolving borax in distilled water and heat the solution to bring the temperature up to that of the oil phase (70–75°C). Slowly add the aqueous phase to the oil phase with continuous stirring to produce an emulsion for the cream base. Mix sandalwood powder or extract in the mixture during stirring. Leave the cream to cool at room temperature with continuous stirring. Store the prepared cream in a cool, dry place.

Final Observation

• Color: Off-white to pale yellow
• Texture: Smooth, uniform, and homogeneous
• Type: Oil-in-water (O/W) emulsion cream

RESULTS AND DISCUSSION

Organoleptic evaluation of formulated cream

Sr. no.	Evaluations Parameters	Results
1	Physical state	Semisolid
2	Odour	Characteristic
3	Colour	Yellowish Brown
4	Consistency	smooth
5	Type of Emulsion	(O/W)
6	Homogeneity	Uniform and homogeneous

pH of the cream

 the range of pH 6.0 to 6.35. These values indicate that the formulation is well suited for the skin since the pH values are similar to those of natural skin.

Sr. No.	Formulation	pH
1	F1	6.00
2	F2	6.22
3	F3	6.20

Viscosity

Sr. No.	Formulation	Viscosity
1	F1	596
2	F2	650
3	F3	645

The viscosity of the cream was found to be in the range of 500–1000 cps, indicating that it can be easily spread with minimal application of shear force.

Spreadability

The range from 13 to 18 g·cm/min, indicating good spreadability with a shorter spreading time.

DPPH Activity –

The solvents tested, the maximum radical scavenging activity observed was 70%.

DPPH Radical Scavenging Activity

Total Phenolic Content

The contents were analyzed in different parts of pomegranate such as peel, flesh, seeds, and whole fruit. The phenolic content was found to increase with more polar solvents. The highest level (67.34 ± 2.77 mg) was recorded in the methanolic extract of the peel. These results agree with Elfalleh et al., who reported higher polyphenol content in methanolic extracts of pomegranate parts.

Antimicrobial Activity

The biological activities of Punica granatum, especially its antibacterial effects, have been extensively studied. In this work, the aqueous extract showed a 22 mm zone of inhibition against E. coli. Its strong antibacterial activity is likely due to its high tannin content (~25%) and other secondary metabolites present in the plant.

Applications

• Helps improve skin firmness and radiance
• Minimizes pigmentation and dark patches
• Enhances the protective effect of sunscreens
• Provides antioxidant and anti-inflammatory benefits
• Inhibits excess melanin formation
• Shields the skin from UV-related damage
• Commonly included in natural creams and gel-based products
• Present in skincare rich in plant-derived polyphenols
• Serves as a safer alternative to synthetic brightening agents

CONCLUSION

Aging of skin is becoming increasingly popular among women and has prompted the need for conducting this study. This research work focused on preparing a natural anti-aging cream that is free from synthetic compounds. Punica granatum (peel extract) was chosen as the principal active agent since it has a high concentration of polyphenols, flavonoids, tannins, and anthocyanins that are responsible for antioxidant and anti-aging properties proven by DPPH assay and total phenol content. Other natural products such as beeswax, borax, almond oil, sandal wood powder, and olive oil were used to make four different formulations using different concentrations. It was found that formulation F1 had excellent stability and smooth texture properties.

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