Ultrasound-Assisted Extraction Optimization and Flash Chromatography Fractionation of Punicalagin from Pomegranate Peel (Punica granatum L.)

Abstract

Background: Pomegranate peel (Punica granatum L.) is a rich source of phenols, particularly ellagitannins, highlighting punicalagin, a bioactive compound with recognized antioxidant potential. However, efficient recovery and purification methods are required to enable its application in food and health-related products. This study aimed to obtain a partially purified fraction of punicalagin from pomegranate peel using optimized extraction and purification strategies. Methods: A Taguchi L9 (3)³ experimental design was employed to optimize ultrasound-assisted extraction, evaluating extraction time (10, 20, 30 min), ethanol concentration (20, 40, 80%), and solid-to-solvent ratio (1:12, 1:14, 1:16). Total polyphenol content was quantified using the Folin–Ciocalteu method. Extracts obtained under optimized conditions were concentrated by rotary evaporation and subjected to semipurification using flash chromatography with Amberlite XAD-16 resin. Subsequently, the fractions were lyophilized and analyzed by HPLC/ESI/MS. Results: The Statistica software determined the optimal conditions for polyphenol extraction (20 min, 40% ethanol, 1:12), with the signal-to-noise (S/N) ratio reaching 88.43 ± 0.66, surpassing the predicted value of 77.42. Flash chromatography yielded four fractions, and HPLC/ESI/MS analysis revealed the presence of ellagitannins in all of them, with fraction number 2 showing the highest relative abundance of punicalagin (89.25%). Conclusions: The combination of ultrasound-assisted extraction and flash chromatography proved effective for obtaining punicalagin-rich fractions from pomegranate peel, supporting its potential for nutraceutical applications.

1. Introduction

The fruit Punica granatum L., commonly known as pomegranate, is native to Asia and the Mediterranean region. It is currently cultivated in North and tropical Africa, North and South America, and the Caucasus area [1]. The fruit is characterized by its nutritional benefits and therapeutic effects due to the high content of bioactive compounds present in the juice, seeds, leaves, flowers, and peel [2].

Pomegranate peel accounts for 43–50% of the total fruit weight and is considered an agro-industrial residue during pomegranate juice production, generating approximately 1.9 million tons of waste worldwide [3,4]. The peel is rich in many polyphenolic compounds, among which ellagitannins stand out, specifically punicalagin, punicalin, and ellagic acid [5].

Punicalagin is the most abundant polyphenolic compound in pomegranate peel. It is an ellagitannin, a type of hydrolyzable tannin, with a molecular weight of 1084.7 g/mol [6]. It is water-soluble and naturally occurs in two anomeric forms, α and β [7]. The literature reports that punicalagin could exert various positive health effects, primarily antioxidant, anti-inflammatory, and anticancer activities, among others [8,9,10]. Moreover, beneficial effects have also been reported on chronic metabolic conditions such as cardiovascular diseases, diabetes, and obesity [11,12,13,14]. These findings highlight the great potential of this molecule for applications in the pharmaceutical and food industries.

Several extraction methods have been reported in the literature to obtain crude extracts rich in polyphenolic compounds from pomegranate peel, using both conventional techniques such as maceration and infusion, as well as advanced technologies including ultrasound-assisted extraction, microwave-assisted extraction, supercritical fluids, pressurized liquids, and enzyme-assisted extraction, among others [15,16]. On the other hand, various purification or semipurification techniques have also been employed, such as medium-pressure liquid chromatography (MPLC), high-speed countercurrent chromatography (HSCCC), as well as methods based on conventional liquid chromatography (column chromatography, preparative HPLC, and Semi-preparative HPLC) [17,18,19].

In this context, flash chromatography emerges as an efficient and versatile alternative that, through the combination of liquid phases and the simultaneous use of two detectors ultraviolet (UV) and evaporative light scattering detector (ELSD) allows the purification or semipurification of organic compounds in significantly shorter times compared to conventional liquid chromatography methods. Its usefulness has increased notably with the development of automated systems, which have optimized their performance and expanded their applications in the purification of bioactive compounds [20]. The aim of this work was to extract, purify, and characterize the ellagitannins from pomegranate peel.

2. Materials and Methods

2.1. Raw Material

The Valencia pomegranate variety was collected in the municipality of Nombre de Dios, Durango, Mexico, from a private orchard located in the town of Cardenchos (latitude 23°51′ N, longitude 104°15′ W, altitude 1755 m above sea level) during the May season by M.C. Karina Guadalupe Reyes-Morales. The fruit components were manually separated, and the pomegranate peels were placed in a forced-air tray dryer at 60 °C for 48 h [21,22]. Subsequently, the dried peels underwent two grinding stages due to the hardness of the material: an initial grinding with an industrial drum mill followed by a secondary grinding with a blade mill (IKA MF-10, Wilmington, NC, USA). The resulting powder was then sieved to obtain a particle size of 500 µm (Humboldt sieve no. 35) and finally stored in vacuum-sealed plastic bags in the dark at room temperature.

2.2. Extraction Bio-Compounds of Pomegranate Peel by Taguchi Methodology

In this study, the Taguchi methodology was used to optimize the polyphenol extraction process from pomegranate peel. An L9 (3³) orthogonal array was applied, testing the three main factors: time (min), ethanol concentration (%), and mass/solvent ratio (

Table 1. Factors and levels for the extraction of phenolic compounds from pomegranate peel.

Factor	Levels
	1	2	3
Time (min)	10	20	30
Ethanol (%)	20	40	80
Mass/solvent ratio	1:12	1:14	1:16

). This array allowed for 9 experimental runs in which the 3 factors were combined at their respective levels (

Table 2. Experimental matrix of the Taguchi l9 (3³) design for ultrasound-assisted extraction of phenolic compounds from Pomegranate peel.

Treatment	Time	Ethanol	Mass/Solvent Ratio
1	1	1	1
2	1	2	2
3	1	3	3
4	2	1	2
5	2	2	3
6	2	3	1
7	3	1	3
8	3	2	1
9	3	3	2

) [23]. After performing the extractions, the total polyphenol content was evaluated for each treatment, and all treatments were carried out in triplicate.

The data obtained were analyzed using Statistica 7.0 software (StatSoft, St. Tulsa, OK, USA) by applying the “higher the better” function. The signal-to-noise ratio (S/N) was used as an indicator of treatment reproducibility and quality, and was calculated using the following equation:

$$\mathrm{S}/\mathrm{N}=-10\mathit{Log}10({\displaystyle \frac{1}{N}}\ast \sum ({\displaystyle \frac{1}{y^2}}))$$

In this equation, the factor −10 ensures that the ratio reflects the inverse of “bad quality,” where y represents the experimental value obtained in each trial and N is the total number of samples.

The analysis of variance (ANOVA) was used to determine the percentage contribution of each factor, calculated as follows:

$$P={\displaystyle \frac{SSi}{SST}}\ast 100\%={\displaystyle \frac{SSi-MSi\ast dfi}{SST}}\ast 100\%$$

where P represents the percentage of contribution, SSi is the individual sum of squares, SST is the total sum of squares, MSi is the individual mean square, and dfi represents the individual degrees of freedom [24].

2.3. Ultrasound-Assisted Extraction

Polyphenol extraction from pomegranate peel was carried out using an ultrasonic bath (model 3800, BRANSON brand, Brookfield, CT, USA) at a frequency of 40 kHz, following the conditions and treatments specified in Table 1 and Table 2, with each treatment performed in triplicate. After extraction, the samples were filtered using Whatman No. 1 filter paper [25].

2.4. Total Polyphenol Content (Folin–Ciocalteu)

The extracted samples were diluted at a 1:100 ratio. To initiate the reaction, 125 µL of the sample was placed in a test tube, followed by the addition of 500 µL of distilled water and 125 µL of Folin–Ciocalteu reagent. The reaction was incubated for 6 min, protected from light and at room temperature. Then, 1250 µL of 7% Na₂CO₃ solution and 1000 µL of distilled water were added. The reaction was incubated again in the dark and at room temperature for 90 min. Absorbance was measured at 760 nm using a spectrophotometer (Genesys 10S UV-Vis Thermo Scientific, Waltham, MA, USA). For the result expression, a calibration curve was prepared from a gallic acid stock solution ranging from 100 to 0 mg/L. The results were expressed as mEq of gallic acid/L [26].

2.5. Flash Chromatography and Semipurification of Ellagitannins

For the fractionation of the polyphenolic extract obtained from pomegranate peel, a method for the semipurification of ellagitannins was developed using a Flash Pure C-850 liquid chromatograph equipped with a binary pump, a UV–visible detector, a light scattering detector (ELSD), and an EcoFlex column (40 g) packed with Amberlite XAD-16. An injection volume of 10 mL was used. The eluents were water (solvent A) and 96% ethanol (solvent B), and the elution of polyphenolic compounds was monitored at 254, 280 and 365 nm. The following gradient was applied: 0% B (linear), 0–5 min; 5% B, 5–6 min; 5% B (linear), 6–16 min; 20% B (linear), 16–17 min; 20% B (linear), 17–22 min; 20% B (linear), 22–24 min; 100% B (linear), 24–27 min; 100% B (linear) [27].

2.6. Lyophilization of Polyphenols

The fractions recovered in 50 mL Falcon tubes were frozen and subsequently lyophilized using a freeze dryer (Labconco Corporation, Kansas City, MO, USA) at a vacuum pressure of 0.340 mBar and a temperature of −49 °C, in order to preserve sample stability of the recovered compounds [28,29].

2.7. Characterization of Polyphenolic Compounds by HPLC/ESI/MS and Quantification of Punicalagin

The characterization of the polyphenolic compounds present in the pomegranate peel extract and in the four fractions recovered after fractionation was performed by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). The analyzed samples were filtered using 0.45 µm nylon membranes and placed in 2 mL vials. High-performance liquid chromatography (HPLC) analyses in reverse phase were performed on a Varian HPLC system, which included an autosampler (Varian ProStar 410, Palo Alto, CA, USA), a ternary pump (Varian ProStar 230I, Palo Alto, CA, USA), and a PDA detector (Varian ProStar 330, Palo Alto, CA, USA). Additionally, an ion-trap mass spectrometer (Varian 500-MS IT Mass Spectrometer, Palo Alto, CA, USA) equipped with an electrospray ionization (ESI) source was used. A 10 µL sample was injected into a Denali C18 column (150 mm × 2.1 mm, 3 µm, Grace, Palo Alto, CA, USA), with the oven temperature maintained at 30 °C. Formic acid (0.2%, v/v; solvent A) and acetonitrile (solvent B) were used as eluents, applying the following gradient: initial, 3% B; 0–5 min, 9% B linear; 5–15 min, 16% B linear; 15–45 min, 50% B linear. Subsequently, the column was washed and reconditioned. The flow rate was maintained at 0.2 mL/min, and elution was monitored at 245, 280, 320, and 550 nm. The entire effluent (0.2 mL/min) was injected into the mass spectrometer source without splitting. All MS experiments were performed in negative mode. Nitrogen [M-H] was used as the nebulizer gas, and helium served as the damping gas. Ion source parameters included a spray voltage of 5.0 kV, with capillary voltage and temperature set to 90.0 V and 350 °C, respectively. Data were collected and processed using MS Workstation software (V 6.9). Samples were analyzed in full-scan mode, acquired over an m/z range of 50–2000. MS/MS analyses were performed on a series of selected precursor ions [30].

3. Results and Discussion

3.1. Total Polyphenol Content from Pomegranate Peel Extracts

In the present study, the Folin–Ciocalteu assay was used to optimize the extraction of phenolic compounds from pomegranate peel through ultrasound-assisted extraction. The Taguchi orthogonal array allowed the exploration of extraction conditions while reducing the number of experiments to nine effective treatments. Figure 1 shows the total polyphenol content, highlighting treatments 2, 5, 6, and 8 for their high content and reproducibility. A statistical analysis of means comparison using Tukey’s test (p < 0.05) showed no significant differences among treatments, indicating that none exhibited statistical superiority. However, since the main objective of the Taguchi design is to identify the combination of factors that maximizes the system response, the signal-to-noise ratio was used to evaluate the effect of each parameter and establish the optimal extraction conditions.

3.2. Relative Influence of Individual Factors

Figure 2 shows the relative influence of individual factors on the extraction of polyphenols from pomegranate peel. Ethanol is observed to be the most decisive factor, contributing 82.97%, followed by the mass-to-volume ratio at 12.20%. Extraction time has a minor impact, accounting for only 1.83%, while the margin of error is 3% (

Table 3. Analysis of variance (ANOVA).

Factors	SS	df	MS	F	p	Percent (%)
Time	1.82	2	0.91	0.61	0.62	1.83
Ethanol	82.49	2	41.24	27.67	0.03	82.97
Mass/Volume ratio	12.13	2	6.06	4.07	0.20	12.20
Error	2.98	2	1.49			3.00
Total	99.42	8				100.00

). These results indicate that solvent selection is the key factor for optimizing the extraction of the target compounds, as it influences the solubility and release of polyphenols from the plant matrix [31]. Its high influence (82.97%) suggests that the efficiency of the process largely depends on the concentration and solvent properties of ethanol. The mass-to-volume ratio (12.20%) also plays an important role, as a greater amount of material in a fixed volume may affect the availability of compounds for dissolution [32]. Extraction time (1.83%) has a negligible effect, suggesting that prolonging the process does not significantly improve compound recovery. The margin of error (3%) reflects experimental variations.

3.3. Optimal Conditions for the Extraction of Total Polyphenol Content

Figure 3 shows a main effects analysis of the signal-to-noise ratio (S/N), used to evaluate the extraction efficiency of polyphenols. It was observed that extraction time at level 2 (20 min), ethanol concentration at level 2 (40%), and the mass-to-volume ratio at level 1 (1:12) were the combinations that offered the best response in terms of S/N. Under these conditions, the software predicted an S/N value of 77.43 (

Table 4. Predicted values and experimental results of total polyphenol content.

Factors	Levels	Value	Effect Size	Standard Error
Time	2	20 min	0.43	0.70
Ethanol Concentration	2	40%	3.15	0.70
Mass/Volume ratio	1	1:12	1.50	0.70
S/N prediction			77.43
S/N experimental validation			88.43 ± 0.66
Polyphenol content (mEq of GA/L.)			8801.89 ± 621.41

). After performing the extraction under the optimal conditions and experimentally validating the process, an S/N value of 88.43 ± 0.66 (8801.89 ± 621.41 mEq of GA/L) was obtained, exceeding the value predicted by the model.

This result indicates a high concentration of total polyphenols in the extract and demonstrates the notable reproducibility of the method. Furthermore, the optimal factor levels found are consistent with literature reports, which suggest that ethanol concentration should be between 40 and 60%, the mass-to-solvent ratio between 1:10 and 1:30, and extraction time between 20 and 40 min to maximize punicalagin yield [33].

3.4. Flash Chromatography and Semipurification of Ellagitannins

The chromatogram obtained during the fractionation stage using flash chromatography shows the separation of four fractions composed of molecules present in the pomegranate peel extract. Most peaks likely correspond to ellagitannins and other polyphenols, as these are characteristic compounds of this plant matrix (Figure 4) [34]. There are some reports in the literature where flash chromatography has been used for the separation of polyphenolic compounds from pomegranate peel with EcoFlex C18 and RediSep C-18 columns; however, the use of Amberlite XAD-16 in flash chromatography for the separation of these compounds has not been previously reported in the literature [35,36]. This resin, employed in adsorption chromatography, favors the retention of polar and hydrophobic compounds such as ellagitannins and other polyphenols, facilitating their separation during the process [37].

3.5. Characterization of the Compounds Present in the Fractions Obtained

As shown in

Table 5. Compounds present in the fractions obtained by flash chromatography.

Peak No.	Compound	Wavelength	RT (min)	[M − H]− m/z	MS2 Ion Fragment (m/z)	F1	F2	F3	F4
1	Gallic acid 4-O-glucoside	273, 380	7.04	330.9	169, 271	-	-	+	-
2	Punicalin	285, 388	8.54	780.6	721, 602, 601	-	+	-	-
3	Pedunculagin I	260, 388	13.52	782.6	481, 375, 301	-	-	-	-
4	Punicalagin α	280, 388	15.64	1083.3	781, 601	-	+	+	-
5	Punicalagin β	280, 388	16.92	1083.3	781, 601	-	+	+	-
6	Ellagic acid derivate	265	21.74	798.6	781, 479, 331, 301, 299	+	+	+	+
7	Granatin B	365, 274	23.88	951.9	934, 933, 915	-	-	-	+
8	Ellagic acid	254, 373	25.40	300.8	301, 229, 185	-	-	+	+

F1: Fraction 1, F2: Fraction 2, F3: Fraction 3, and F4: Fraction 4.

, the molecules present in the fractions are mainly ellagitannins, which is consistent with reports in the literature stating that ellagitannins are the most abundant phenolic compounds in pomegranate peel [38]. Furthermore, other authors have reported a similar compound profile after a semipurification process using Amberlite XAD-16, indicating that ellagitannins have a high affinity for this chromatographic resin [39].

Fraction number two (Figure 5) showed the highest punicalagin content with 89.25% and the presence of only ellagitannins (

Table 6. Characterization of pomegranate peel ellagitannins present in Fraction 2.

No.	Time (min)	[M − H]−	Compound
1	17.80	780.6	Punicalin
2	18.70	1082.3	Punicalagin α
3	19.86	1082.3	Punicalagin β
4	21.21	798.7	Ellagic acid derivate

). From 191 g of dry matter (pomegranate peel), 1.5 g of this fraction was obtained, corresponding to a yield of 0.78%. Previous studies have identified punicalagin α and β as the main compounds after purification processes using UHPLC [40]. Purifications performed by mass-directed semi-preparative ESI-AP single quadrupole LC-MS have achieved punicalagin recoveries with purities above 95%, while preparative HPLC has reported purities up to 98.05% [41,42]. Nevertheless, in this study, a considerable concentration of punicalagin was obtained using flash chromatography, a more accessible, faster, and scalable technique that operates at high flow rates and low pressure. This high concentration reflects the strong selectivity of the chromatographic system, enabling the recovery of an enriched fraction without resorting to more expensive purification methods, thus supporting the feasibility of its industrial application [43].

4. Conclusions

The extraction of phenolic compounds from pomegranate peel was successfully carried out, demonstrating the effectiveness of both the experimental design and the extraction method used. The crude extract was efficiently fractionated using flash chromatography, yielding four fractions rich in hydrolyzable tannins. Furthermore, characterization by HPLC/ESI/MS confirmed the presence of ellagitannins in all four fractions, with fraction number two standing out due to its high punicalagin content. These results support the potential of pomegranate peel as a valuable source of ellagitannins, with promising applications in the pharmaceutical and food industries.