Bioactive compounds of Punica granatum L. wastes by high performance liquid chromatography analysis

Abstract The massive pomaces of Punica granatum L. exhibit a challenging losses exposure difficulty for the processing industries. The resent study was aimed to investigate the bioactive compounds of pomace extracts to introduce it to different industries such as pharmaceutical, food, medicinal, agricultural etcetera for optimum use. Four different extracts were prepared and the phenolic compounds were quantified using HPLC-DAD. Different amounts of phenolic compounds were detected in the samples including gallic acid, catechin, ellagic acid, rosmarinic acid, hesperidin, p-coumaric acid and chlorogenic acid. Gallic acid was major compound in all studied extracts of pomaces, with the maximum amount belonging to water extract (at 60 °C). The average amount of gallic acid detected in water extract (at 60 °C) of Punica granatum L. was 11.25 mg g−1 dry weight, while it was 3.24 3.02 and 1.09 mg g−1 dry weight for the extracts obtained by distilled water, methanol and methanol 80%, respectively. Graphical Abstract


Introduction
One of the family Lythraceae, subfamily Punicoideae which has been used as an edible fruit since antiquity, is pomegranate (Punica granatum L.). It is a native plant that greatly dispensed in south of Iran. Pomegranate is a source of antioxidants because of the presence of phenolic and tannin compounds (Loren et al. 2005). It is used in medicinal, food and cosmetic formulations (Finkel and Holbrook 2000) and can be introduced as a well source of antioxidants (Singh et al. 2001). From epochal to recent times, different parts of pomegranate have been used for different objects such as in diets (e.g. juices, jams, jellies, dressings, marinating, and wine), or as religious symbolism (e.g. righteousness, fullness, fertility, abundance), or for its medicinal values. High nutrient composition such as, oxalic acid, potassium, folate and vitamins E, C, B 6 and A is well demonstrated in pomegranate peels (Al Rawahi et al. 2014). Generally, low aromatic intensity is the main characteristic of pomegranate fruit (Wang et al. 2013). A hydroquinone pyridinium alkaloid from the leaves (Schmidt et al. 2005), punigratane, as a pyrrolidine alkaloid from the rind and with its efflux inhibition activity (Rafiq et al. 2016), tricetin 4 0 -O-b-glucopyranoside as a flavone glucoside and four ellagitannins and flavones (tricetin, luteolin, ellagic acid, and granatin B) from the flowers (Wu and Tian, 2019) of pomegranate were isolated. Moreover, antioxidant, antimicrobial, antidiabetic potential, antiparasitic activities and a-glucosidase and maltase inhibitory effects of pomegranate leaf, rind and flower extracts have been illustrated (El Dine et al. 2014;Rahmani et al. 2017;El Deeb et al. 2021). Previous studies have been on different parts of the healthy fruit of the plant after harvest without the fruit being mechanically pressed by the juicer. No study was observed under our conditions on the rest of the plant which is abandoned and discarded in factories after the dewatering step. As most parts of the fruit including exocarp, endocarp, pulp, stems and seeds that are not edible, it is not as popular as other family members. They are discarded as wastes in the environment. On the other hand, the value of these wastes is not well known. So, the investigation of phenolic compounds of pomace of pomegranate by HPLC-DAD was our aim to be able to introduce it to various industries for more use and application.

HPLC validation
The R 2 quantities from calibration curves of standard phenolic compounds were in the span from 0.985 to 0.999 which confirmed the linearity of the method. The RSD values for the accuracy studies were below 2.0%. The HPLC method was precise in the quantitative analysis of phenolic compounds.

Phenolic composition
The variance analysis illustrated significant differences in phenolic compounds among the different extracts in P. granatum (P < 0.01; Table S1). Our findings illustrated that gallic acid was the abundant phenolic compound in all studied pomegranate pomace extracts (PPEs) and it was agreed with other previous studies. According to the previous reports gallic acid is the main phenolic compounds in peel extract of pomegranate. The pomaces included the peels, also. Gallic acid and ellagic acid may be the compounds responsible for P. granatum anti-inflammatory effect. Hydrolyzable tannins have a polyhdric alcohol at their core, the hydroxyl groups of which are partially, or fully, esterified with either gallic acid or ellagic acid. They may have long chains of gallic acid coming from the central glucose core. On hydrolysis with acid or enzymes, the hydrolyzable tannins break down into their constituent phenolic acids and carbohydrates. As a result, Al Rawahi et al. (2014) was reported major phenolic compounds in P. granatum peel extract cultivated in Oman as gallic acid, illogic acid, punicalin, and punicalagin. The phenolic acids such as ellagic acid, gallic acid, chlorogenic acid, caffeic acid, vanillic acid, ferulic acids, trans-2hydrocinnamic acid and quercetin in pomegranate are identified (Bassiri-Jahromi and Doostkam, 2019). In our study the greatest content of gallic acid was determined in water extract at 60 C in the water bath. Phenolic compounds have a considerable structural diversity, characterized by the hydroxyl groups on aromatic rings. According to the number of phenol rings and the structural elements that bind rings to one another, such compounds are grouped and classified as simple phenols, phenolic acids, flavonoids, xanthones, stilbenes, and lignans. Phenolic compounds in P. granatum pomace in our study included 2 hydroxybenzoic acids and as hydrolyzable tannins (gallic acid and ellagic acid), 3 hydroxycinnamic acids (rosmarinic, p-coumaric and chlorogenic acids), one flavanon glycoside (hesperidin) and one flavan-3-ol (catechin). During the industrial extraction process, the tannins pass to juice while the high antioxidant capacity of pomegranate is attributed mainly to these compounds. At four PPEs in our present study, gallic acid and ellagic acid, were detected and identified. Caffeic acid was not found in our study. Caffeic acid, is biosynthesized by hydroxylation of coumaroyl ester of quinic acid (esterified through a side chain alcohol). This hydroxylation produces the caffeic acid ester of shikimic acid, which converts to chlorogenic acid. We did not find caffeic acid but the esters of it, rosmarinic acid and chlorogenic acid, were identified in all samples. Also, this can depend on the type of sample. The cultivar, genotypes, extraction methods, etc have a higher influence on the phenolic content. Different pomegranate cultivars had different polyphenol compositions. It is considerably associated with many factors such as cultivar type, growing region, maturity, cultivation, climate, edaphic condition, and storage situation. Rosmarinic acid exhibits antioxidant and anti-inflammatory effects and has recently been shown to protect neurons in vitro against oxygen-glucose deprivation.

Experimental
See supplementary material.

Conclusions
Different amounts of phenolic compounds were detected in the samples including gallic acid, catechin, ellagic acid, rosmarinic acid, hesperidin, p-coumaric acid and chlorogenic acid. Gallic acid was major compound in all studied extracts of pomaces, with the maximum amount belonging to water extract at 60 C in the water bath. According to the findings of this study, the pomaces of P. granatum are natural sources of phenolic compounds. The verified P. granatum pomace and its related bioactive components like flavonoid and phenolic compounds can have a powerful potential as a novel device for inhibiting different human diseases and a chemo prohibitive. Several beneficial effects are reported for these phenolic compounds, including antioxidant, anti-inflammatory, and antineoplastic properties. These compounds have been reported to have therapeutic activities in gastrointestinal, neuropsychological, metabolic, and cardiovascular disorders (Lin et al. 2018). It is estimated that total production amounts to around 3 million tons of pomegranate are produced in the world, annually, of which Iran produces approximately 28%, annual production of pomegranate has been recorded as 10,866,300 tons. After pressing of fruits for juice or oil, the solid remains are pomace. It includes the stems, seeds, pulp and skins of the fruit. During the Punica juice processing, about 40 to 50 percent of the products were retained. It is possible to keep the waste of these crops one hundred thousands of tons were estimated (Animal Science Research Institute of Iran (ASRI), 2015). Whereas desirable utilization of agricultural and food wastes such as pomaces of P. granatum will reduce costs and environmental hazards in which results from their disposal and remaining in the environment, our studies are continuing to investigate and introduce pomaces of P. granatum as strong natural resources and we are going to use these pomaces for other goals.