Screening of Major Bioactive Compounds Profile of Pomegranate Peel Waste by Using HPLC


 Bioactive compound characterization is an important steps for utilization of pomegranate peel waste as food and nutraceutical ingredients. In the present investigation the effects of different drying (freeze, tray-oven and sun) and solvents such as methanol, ethanol, water, acetone and hexane were investigated on the extraction and recovery of major bioactive compounds (ellagic acid, gallic acid, quercetin and punicalagin) of pomegranate peel for two pomegranate varieties (i.e. Bhagwa and Ganesh) using high performance liquid chromatography (HPLC). The results indicated that the peel powder obtained by freeze drying (-45°C) method which extracted by methanol was higher amount in bioactive compounds compared to the other drying methods and solvents. Freeze dried peel powder of Bhagwa pomegranate showed higher amount of gallic acid (32.2 mg/g), ellagic acid (13.6 mg/g), punicalagin (15.2 mg/g) and quercetin (2.5 mg/g) with methanol solvent as compared to the other extract of Bhagwa and Ganesh varieties. Basis on the results of current study it can be concluded that the freeze drying method for drying pomegranate peel powder and methanol as a solvent are effectives to recover higher extraction of bioactive compounds which can be utilize to develop human and animal health.


Introduction
Pomegranate fruit is known as Super fruits due to its delicious taste. It contains approximately 48-50% as waste of total fruits after juice extraction, which corresponds the pomegranate peel waste [1][2] . The pomegranate peel is important source for natural bioactive compounds such as ellagitannins, tannis, gallic acid, punicalagin, catechin, rutinpunicalin, laempferol, luteolin, glycosides and epicatechin among other phenolic compounds [3][4][5][6][7] . Despite that the gallic acid, ellagic acid, punicalagin and quercetin are considered major bioactive compounds of pomegranate peel. These bioactive compounds are responsible for the different biological activities such as antimicrobial, antioxidant, anticancer, antimutagenic, and anti-in ammatory and help to reducing the risk of chronic and cardiovascular diseases 8- 10 .Several researchers have reported the biological activities and functions of pomegranate peel 11-12 . However, the drying methods are very important factors for the drying of products to removing water and reducing the chemical reaction or enzymatic activities [13][14][15][16][17] .The drying methods can affects the quality attributes such as color, nutritional and phytochemical activities of products 10,[18][19][20] . Various types of drying methods such as sun drying, vacuum drying, freezing drying, oven drying and air drying etc. are used to drying the products 21 . Furthermore the freeze drying known as potential method for extraction and higher recovery of bioactive compounds and other phytochemical from natural plant sources compared to other drying methods; however the freeze drying method is expensive method compared to other but retained higher quality of the products 22-25 . In addition, the extraction of bioactive compounds from plant sources is considered as primary steps and the solvents are important factors for the extraction and recovery of bioactive compounds. Several types of solvents i.e. polar and non-polar are used to extract the bioactive and phenolic compounds from plant.
Generally, the non-polar and low polar solvents are used to extract lipophilic compounds and pigments from plant. However, the recovery of phenolic compounds, yield and their free radical scavenging activity, antimicrobial and other biological activities of pomegranate peels and other plants are depending on the types of solvents and extraction procedure 12,[26][27][28][29][30]

Materials
The fresh pomegranate fruits were procured from pomegranate orchard, Kullu, Himachal through National Research Center on Pomegranate (NRCP-ICAR), Solapur, Maharashtra (India). The study complies with local and national guidelines.
Chemical and reagents. The analytical grades of chemicals, reagents and standards purchased from Sigma Aldrich Inc. and Hi-Media, India.
Preparation of peel powder. The pomegranates were peeled manually to obtain the peel. The peel as obtained was subjected to different drying methods to obtain peel powders (PGP). The blanching of obtained fresh pomegranate peel was carried out in water bath at 90°C for 30 sec to remove surface impurities and contamination and dried under three different conditions viz., freeze drying (-45°C for 32 h), tray-oven (60°C for 29 h) and sun dried (72 h) respectively ( Figure 6). Ultrasonic Extraction. The dried peel powder obtained from the peels of Bhagwa and Ganesh was used for the extraction of phenolic and avonoid content using polar (viz., methanol, ethanol, water, acetone) and non-polar solvents (hexane) through ultra-sonic assisted extraction method. The ne powder samples (0.2 g) of pomegranate peel of both varieties were sonicated in 10 ml of different solvents using an ultrasonic bath (CUB-5, Citizen, 40 kHz, 220-240 V, India) for 30 min at 45ºC temperature 10 . The prepared solution was centrifuged (Sigma, 3-18, KS, Germany) at 5ºC for 10min with 8654 RPM and ltered using Whatman No. 11 lter paper to obtain the transparent extract. In total, there were three pomegranate peel powder samples for each variety prepared through three different drying methods (freeze drying, tray drying, and sun drying). For each type of powder, ve solvents (methanol, ethanol, water, acetone, and hexane) were used for preparing extracts. The peel extracts were later used for estimation of total phenolic content, total avonoid content, antioxidant and antibacterial activity. The quanti cation of major phenolic content in the pomegranate peel of both the varieties was also done by HPLC analysis. The 15 type's extract of pomegranate peel powder for each variety was constructed with the completely randomized design (CRD) of research. The coding of extracts as prepared has been presented in Table 2. Origin Pro (2019b) was used to graphically representation of average data.

Results
HPLC chromatogram of standards. The chromatograms of phenolic compounds standards such as ellagic acid, gallic acid, quercetin, and punicalagin in terms of retention time, area and height are shown in Table 1 and Figure Figure 2. The ellagic acid compound was estimated through HPLC and has been expressed as mg/g of peel powder. The results indicated that there was a signi cant impact of speci c solvent used to extract the ellagic acid in both Bhagwa and Ganesh peel powders. Apart from solvents used, there was also a signi cant impact of the method of drying to prepare the peel powders on ellagic acid content. Among various solvents used, extracts prepared with methanol solvent showed the highest ellagic acid content in all the three types of peel powders (freeze dried, tray dried, and sun dried) followed by ethanol, water (control) and acetone in both the pomegranate varieties. The least ellagic acid content was recovered with hexane being used as an extraction solvent. The ellagic acid content recovered using different solvents were also statistically signi cant with respect to each other for a speci c type of drying treatment. In peel powder of Bhagwa, the statistically signi cant and maximum ellagic acid content (32.20±0.01mg/g) was recorded in freeze dried peel powder. This ellagic acid content was also statistically signi cant and highest as compared to all other solvents and drying treatments.
The second best result was obtained with ethanol solvent in freeze dried powder (30.19±0.01mg/g) followed by methanol solvent in tray dried powder (28.40±0.02mg/g). Among various drying treatments, the statistically signi cant and highest ellagic acid content was recovered in freeze dried powder followed by tray dried and sun dried peel powders. The least ellagic acid content was recovered in sun dried peel powder. The ellagic acid content as observed in different drying treatments was also statistically signi cant with respect to each other for a speci c types of solvent. In peel powder of Ganesh, the statistically signi cant and maximum ellagic acid content (17.50±0.02mg/g) was recorded in freeze dried peel powder. This ellagic acid content was also statistically signi cant and highest as compared to all other solvents and drying treatments. The second best result was obtained with ethanol solvent in freeze dried powder (14.98±0.2mg/g). It was also observed that the ellagic acid content was statistically at par for tray dried peel powders with hexane and acetone as solvents (1.30±0.03 mg/g, 1.30±0.01mg/g, respectively). It was also noted that in peel powder of Ganesh variety, irrespective of the drying treatments, the ellagic acid content was comparatively very low in acetone, hexane, and control (water) solvents as compared to methanol and ethanol.
The obtained results showed that methanol and ethanol could be used for the extraction of ellagic acid compounds from peel powder. The freeze drying method and methanol solvent are signi cantly accounted to recover a higher amount of ellagic acid from pomegranate peel as compared to respective drying treatments and solvents. The hexane solvent was not detected to quantify biological compounds from pomegranate peel powder due to lower e ciency to extract high polar compounds 17,39 .
Gallic acid. The phenolic compound such as gallic acid passes a wide spectrum of biological activities, which include phenolic, antioxidant, and antibacterial activities, etc. 40 . The results pertaining to extraction of gallic acid content from three different types of pomegranate peel powders obtained from Bhagwa and Ganesh varieties have been presented in Figure 3. The results indicated that there was a signi cant impact of speci c solvent used to extract the gallic acid bioactive compound in both Bhagwa and Ganesh peel powders. Apart from solvents used, there was also signi cant impact of the method of drying to prepare the peel powders on gallic acid. Among various solvents used, extracts prepared with methanol solvent showed the highest amount of gallic acid in all the three types of peel powders followed by ethanol, water, and acetone in both the pomegranate varieties. The least gallic acid content was recovered with hexane being used as an extraction solvent. The gallic acid bioactive compound recovered using different solvents were also statistically signi cant concerning each other for a speci c type of drying treatment.
In peel powder of Bhagwa, the statistically signi cant and maximum gallic acid content (16.40±0.02mg/g) was recorded in freeze dried peel powder. This gallic acid content was also statistically signi cant and highest as compared to all other solvents and drying treatments. The second best result was obtained with ethanol solvent (16.20±0.10mg/g) followed by water as a extraction solvent in freeze dried powder (13.60±0.02mg/g). Among various drying treatments, the statistically signi cant and highest gallic acid content was recovered in freeze dried powder followed by tray and sun dried peel powders. The least gallic acid content was recovered in sun dried peel powder. The gallic acid content as observed in different drying treatments was also statistically signi cant with respect to each other for a speci c type of solvent. The least recovery of gallic acid from peel powder of Bhagwa pomegranate was found with hexane solvent (5.40±0.01mg/g) in freeze dried followed by tray dried (0.80±0.02mg/g) respectively. The peel powder obtained by sun drying treatment was not found effective for recovering gallic acid compounds with hexane solvent. In peel powder of Ganesh, the statistically signi cant and maximum gallic acid content (10.90±0.03mg/g) was recorded in freeze dried peel powder. This amount of gallic acid content was also statistically signi cant and highest as compared to all other solvents and drying treatments. The second best result was obtained with methanol solvent in tray dried powder (2.00±0.10mg/g). It was also noted that in peel powder of Ganesh variety, irrespective of the drying treatments, the gallic acid content was comparatively very low in ethanol, water, acetone, and hexane solvents as compared to methanol. The peel powder extraction with hexane solvent showed gallic acid compound in freeze dried (0.80±0.02mg/g). In peel powder obtained from tray and sun drying treatments with hexane solvent not detected gallic acid compounds. In peel powder obtained from tray drying treatment also not recovers gallic acid with acetone as solvent. The results pertained that the maximum gallic acid was obtained with methanol as a solvent followed by ethanol in both the pomegranate varieties. The results also indicated that peel powders of Bhagwa variety recovered signi cantly higher amount of gallic acid as compared to Ganesh. The results demonstrated that the freeze drying method is signi cant potential to recovering the higher amounts of gallic acid as compared to other drying methods 33 .
Quercetin. The results of quanti cation of quercetin from pomegranate peel powders are shown in presented in Figure 4. The results indicate that there is a signi cant impact of speci c solvent used to extract the quercetin in both Bhagwa and Ganesh peel powders. Apart from solvents used, there was also a signi cant impact of the method of drying to prepare the peel powders on quercetin content. Methanol as a solvent showed the highest amount of quercetin, followed by ethanol, water, and acetone solvents in both the Bhagwa and Ganesh varieties. The peel powder obtained from freeze drying was showed the statistically signi cant and highest amount of quercetin followed by tray and sun dried peel powder obtained from both the Bhagwa and Ganesh varieties. In peel powder of Bhagwa, the statistically signi cant and maximum quercetin (2.50±0.01mg/g) was estimated in freeze dried peel powder. This quercetin was also statistically signi cant and highest as compared to all other solvents and drying treatments.
The second best result was obtained with ethanol solvent in freeze dried powder (1.40±0.01mg/g) followed by water solvent in freeze dried powder (1.10±0.10mg/g). Among various drying treatments, the statistically signi cant and highest quercetin content was recovered in freeze dried powder followed by tray dried and sun dried peel powders. The least quercetin content was recovered in sun dried peel powder. The quercetin content as observed in different drying treatments was also statistically signi cant with respect to each other for a speci c type of solvent. The peel powder extraction with hexane were not quanti ed the quercetin compounds in all drying treatment conditions. Quercetin compound was not detected with acetone solvent in in peel powder obtained from sun drying method.
In peel powder of Ganesh, the statistically signi cant and maximum quercetin compound (0.53±0.01mg/g) was recorded in freeze dried peel powder. This quercetin was also statistically signi cant and highest as compared to all other solvents and drying treatments. The second best result was obtained with methanol solvent in tray dried peel powder (0.52±0.58mg/g). It was also noted that in peel powder of Ganesh variety, irrespective of the drying treatments, the quercetin compound was not detected in acetone and hexane solvents. The peel powder obtained from tray and sun drying treatments was also not showed the quercetin compounds with water solvent. Overall results showed that the maximum quercetin was obtained with methanol as solvent followed by ethanol in both the pomegranate varieties. The results also indicated that peel powders of Bhagwa variety had signi cantly a higher recovery of quercetin as compared to Ganesh. The methanol and ethanol solvent are accounted for suitable solvents for the extraction of quercetin compound as compared to other solvents due to higher polarity and e ciency 41 .
Punicalagin. The results pertaining to extraction and recovering of punicalagin compound from pomegranate peel powders are shown Figure 5. The punicalagin compound was estimated through HPLC and has been expressed as mg/g of peel powder. The results indicated that there was a signi cant impact of speci c solvent used to extract the punicalagin in both Bhagwa and Ganesh peel powders. Apart from solvents used, there was also a signi cant impact of the method of drying to prepare the peel powders on punicalagin. Among various solvents used, extracts prepared with methanol solvent showed the highest punicalagin in all the three types of peel powders (freeze dried, tray dried and sun dried) followed by ethanol, water, and acetone in both the pomegranate varieties.
The least amount of punicalagin was recovered with acetone and hexane being used as an extraction solvent. The punicalagin compound recovered using different solvents were also statistically signi cant with respect to each other for a speci c type of drying treatment. In peel powder of Bhagwa, the statistically signi cant and maximum amount of punicalagin compound (15.20±0.20mg/g) was recorded in freeze dried peel powder. This punicalagin compound was also statistically signi cant and highest as compared to all other solvents and drying treatments. The second best result was obtained with ethanol solvent in freeze dried powder (13.80±0.02mg/g) followed by methanol solvent in tray dried powder (11.20±0.02mg/g). Among various drying treatments, the statistically signi cant and highest punicalagin compound was recovered in freeze dried powder followed by tray dried and sun dried peel powders. The least punicalagin compound was recovered in sun dried peel powder. The punicalagin compound as observed in different drying treatments was also statistically signi cant with respect to each other for a speci c type of solvent. The peel powder obtained from all respective drying (freeze drying, tray drying, and sun drying) methods were not recovered punicalagin compound with acetone and hexane solvent respectively. In peel powder of Ganesh, the statistically signi cant and maximum recovery of punicalagin compound (7.30±0.01mg/g) was recorded in freeze dried peel powder. This punicalagin content was also statistically signi cant and highest as compared to all other solvents and drying treatments. The second best result was obtained with ethanol solvent in freeze dried powder (4.60±0.02mg/g). The similar trend of results was also observed in peel powder obtained from tray drying method. It was also noted that the peel powder obtained from freeze drying and tray drying method was not recovered punicalagin compound with acetone and hexane solvent respectively. In sun dried peel powder extraction was also not recovered the punicalagin compound with water, acetone and hexane solvent due to lower e ciency to extract phenolic compounds from pomegranate peel. the results of present study are in line with previous study done by Singh et al.,38 ; those reported higher recovery of punicalagin compound with methanol as solvent followed by ethanol in both the pomegranate varieties.
The results also indicated that peel powders of Bhagwa variety had a higher recovery amount of punicalagin compound as compared to Ganesh. The results demonstrated that the freeze drying method and methanol solvent for extraction was signi cant potential to retaining and extraction the higher amounts of punicalagin content as compared to other drying methods and solvents.
In Summary, the Bhagwaextract exhibited the greatestquantity of phenolics such as gallic acid, punicalagin, quercetin and ellagic acid compared withGaneshextract. Methanolic extract exhibited the greatestamount of phenolicssuch as ellagic acid, gallic acid and Punicalagin. The highest quantity of gallic acid was detectedforBhagwa in aqueous solvent. The study reported that hexane and acetone solvents are not suitable for the phenolic extractions in pomegranate peels. Overall the freezing drying method and methanol as extraction solvent for extraction bioactive compounds from pomegranate peel are highly recommended. Further studies are needed to explore to check the e ciency of combination of solvents (polar/non-polar) for higher recovery of natural bioactive compounds from pomegranate peel waste and other natural sources for further application in food and pharmaceutical sectors at commercial scale.

Discussion
The freeze drying method and methanol solvent are signi cantly accounted to recover a higher amount of ellagic acid from pomegranate peel as compared to respective drying treatments and solvents. The hexane solvent was not detected to quantify biological compounds from pomegranate peel powder due to lower e ciency to extract high polar compounds 17,39 . The results have showed that the maximum gallic acid was obtained with methanol as a solvent followed by ethanol in both the pomegranate varieties. The results also indicated that peel powders of Bhagwa variety recovered signi cantly higher amount of gallic acid as compared to Ganesh. The results demonstrated that the freeze drying method is signi cant potential to recovering the higher amounts of gallic acid as compared to other drying methods 33 . Quercetin phenolic content was signi cantly higher obtained with methanol as solvent followed by ethanol in both the pomegranate varieties. The results also indicated that peel powders of Bhagwa variety had signi cantly a higher recovery of quercetin as compared to Ganesh. The methanol and ethanol solvent are accounted for suitable solvents for the extraction of quercetin compound as compared to other solvents due to higher polarity and e ciency 41 . In case of punicalagin compounds, the sun dried peel powder was not potential to extract punicalagin compounds with water, acetone and hexane solvent due to lower e ciency to extract phenolic compounds from pomegranate peel.  17 . They reported the drying method had a direct impacts on the recovery of bioactive compounds from pomegranate peel. The freezing drying is most desirable method to retain the higher amount of bioactive compounds such as ellagic acid, gallic acid, quercetin and punicalagin of pomegranate peel. They also con rmed that the methanol as extract solvent is more potential to recover higher amount of gallic acid, ellagic acid, quercetin and punicalagin content from pomegranate peel powder due to high polar nature of methanol. The non-polar solvent such as hexane is not potential to recovering the bioactive contents from pomegranate peel powders.  Different dried pomegranate peel powders Where; BFD= Bhagwa freeze dried, BTD= Bhagwa tray dried, BSD= Bhagwa sun dried, GFD=Ganesh freeze dried, GTD= Ganesh tray dried, GSD= Ganesh sun dried