Polyphenolic profile and antioxidant properties of Momordica charantia L. ‘Enaja’ cultivar grown in Romania

Abstract This is the first study describing phenolics of Momordica charantia L. ‘Enaja’ cultivar (bitter melon) produced in Romania. Total polyphenol content, total tannin content, total flavonoid content, and antioxidant activity of bitter melon stems and leaves, young fruits, and ripe fruits grown in Romania were analysed, along with fruits imported from India. The UPLC-DAD analysis led to the identification of (+)-catechin, (−)-epicatechin, luteolin-3’,7-di-O-glucoside, luteolin-7-O-glucoside and vanillic acid. (−)-Epicatechin (859 µg/g) and (+)-catechin (1677 µg/g) were the most abundant compounds in stems and leaves, while in the ripe fruits, luteolin-7-O-glucoside (310 µg/g) was the main phenolic. Stems and leaves were the most active for capturing free DPPH radicals (IC50 = 216.9 ± 11.91 µg/ml); the scavenging activity strongly correlated with the flavonoid content (r = 0.8806, r2 = 0.7754). Momordica charantia fruits from Romania, both young and ripe, are a source of polyphenols as valuable as those imported from India. Graphical Abstract


Introduction
Momordica charantia L. (mC) (Cucurbitaceae), commonly called bitter melon, bitter gourd, or balsam pear, is a tendril-bearing vine grown in tropical areas of asia, East africa, amazon, and the Caribbean for its edible fruit.it is now widely distributed throughout tropical and subtropical regions and has been reported as invasive in the pacific, Brazil, and the Caribbean (CaBi 2014).the fruit is consumed as a vegetable by asian populations throughout the world and is also a popular folk medicine in China, india, Sri Lanka, and Central america. in addition to its major use as an anti-diabetic agent, bitter melon is used in ethnomedicine as a tonic, emetic and laxative (nagarani et al. 2014a;Rahman et al. 2015;Huang et al. 2018).Bitter melon comes in a variety of shapes and sizes.the typical indian varieties, have a narrower shape with pointed ends and have a tuberculate surface presenting numerous swellings.in Romania, several varieties are grown, such as 'Brâncuşi' , 'Rodeo' , or 'Enaja' .'Enaja' cultivar fruits are 10-20 cm long, with a wart-like green exterior, when fully ripe, the fruits turn to orange and splits into segments, and the seeds covered by red aril can be seen.the fruits are harvested when the peel is dark green and the pulp as well as the seeds are soft and white.unlike these, the common Chinese variety is oblong, 20-30 cm long, and pale green with a gently undulating surface.the vein (stem and leaves) and fruits of bitter melon are used in food supplements that are sold as anti-diabetic (peter et al. 2019) and anti-obesity (Fan et al. 2021) remedies.other medicinal claims are also attributed to different organs of the plant, like bitter stomachic, antiviral, antibacterial, and anticancer activity (nagarani et al. 2014a;Huang et al. 2018;Zubair et al. 2018;Sur et al. 2020).
Besides the huge number of in vitro and in vivo investigations, also several controlled trials were performed with bitter melon to assess its hypoglycaemic effect in diabetes mellitus type 2. in randomised controlled trials (RCts) preparations obtained from unripe green fruits (trakoon-osot et al. 2013; peter et al. 2020), sometimes only the fruit pulps or juice of the fruits (Rahman et al. 2015) were used.Chemical markers used in quality control of the products used in trials were charantin (Zanker et al. 2012;trakoon-osot et al. 2013;Rahman et al. 2015) or uridine (Suthar et al. 2016), but some trials did not include the quantification of some specific substances (Krawinkel et al. 2018).
the main phytochemicals which can be related to the pharmacological effects of bitter melon are the cucurbitane-type triterpenoids (Harinantenaina et al. 2006;Wang et al. 2012;Zeng et al. 2014;alam et al. 2015;Jia et al. 2017;Venkatakrishnan et al. 2019), phenolics (Horax et al. 2006), poly-peptides (Choudhary et al. 2012), carotenoids (Saini et al. 2017).polyphenols are a class of phytochemicals widely known for their positive role in maintaining health, and certain dietary polyphenols can influence carbohydrate metabolism at several levels (thomas et al. 2012). in Momordica charantia aerial parts Svobodova et al. (2017) identified mainly derivatives of quercetin and kaempferol.Kubola et al. (2008) have shown that the main phenolic compound in leaf, stem, green, and ripe fruits is gallic acid.Regarding different extraction techniques for bioactive antioxidants, such as polyphenolics, Hani et al. (2017) described the optimal extraction conditions using ultrasound-assisted extraction.
Bitter melon's growing popularity in Europe is obvious, with Spain being the leader among Momordica exporters in the world (tridge 2023), and the greatest importer in the world is the uSa followed by France and Germany (tridge).at present, only a small number of investigations have been carried out to evaluate the parameters of bitter melon grown in Europe.Busuioc et al. (2020) assessed the antioxidant properties of juice obtained from M. charantia 'Brâncuşi' , a Romanian variety.Cultivation parameters and preliminary phytochemical analysis were published for the 'Enaja' cultivar grown in Romania (Keseru et al. 2016a(Keseru et al. , 2016b(Keseru et al. , 2018)). in previous work, we assessed the charantin content of the M.charantia 'Enaja' cultivar grown in târgu mureş (Romania) and showed that is comparable to those grown in india (Laczkó-Zöld et al. 2021).this study aims to characterise for the first time the antioxidant activity and phenolic content of the Momordica charantia 'Enaja' cultivar grown in târgu mureş, Romania.phenolic content was also compared to those of a commercial product originating from india.

Results and discussion
in this study, the chemical composition of Momordica charantia 'Enaja' cultivar grown in târgu mureş (Romania) was analysed, with a focus on the polyphenolic substances.Compounds were identified by comparison of their chromatographic characteristics with that of available reference substances, and by the uV-ViS spectra.the concentration of the compounds determined from chromatograms was calculated with the help of the calibration equations given in supplementary material (see table S1 in Supplementary material).the stem and leaf of Momordica charantia showed high (−)-epicatechin concentration (1677.974µg/g) and (+)-catechin (859.39 µg/g). in addition luteolin-7-O-glucoside (598.328µg/g), luteolin-3′,7-di-O-glucoside (383.972µg/g) and vanillic acid (363.323µg/g) were quantified (see table S2). in the ripe fruits from Romania luteolin-3′,7-di-O-glucoside and vanillic acid were quantified, while in the fruits imported from india, only vanillic acid could be measured by upLC-pda (table S2).these compounds have already been found by the scientific community in many other plants of the Momordica genus and Momordica charantia varieties (Horax et al. 2006;Kubola et al. 2008) but had not been previously identified and quantified in the 'Enaja' variety.the compounds (−)-epicatechin, (+)-catechin, and luteolin glucosides found in stems and leaves of Momordica charantia, are related to anti-inflammatory, antioxidant, and anticancer activities (Loke et al. 2008;dower et al. 2015;Kikuchi et al. 2019).Similar differences among the organs' polyphenolic content are considered normal and were also observed earlier.Horax et al. (2006) investigated the phenolic compounds of several bitter melon variants, and very different catechin-epicatechin ratios were found in the samples (from 1:1.6 w/w to 4.6:1 w/w).Kubola et al. (2008) have investigated the phenolic content of various methanol extracts from the stem, leaves, green fruit and ripe fruit of bitter melon.they also found differences among the organs' phenolic compound content.
the stems and leaves presented a higher concentration of total polyphenols (12.36 ± 1.15 mg GaE/g), tannins (0.022 ± 0.0046% pyrogallol), and flavonoids (3.29 ± 0.149 mg QE/g) than the other plant organs, such as young fruits and ripe fruits (see table S3). the fruits of several species belonging to the Momordica genus are an excellent source of phenolic compounds (nagarani et al. 2014a).the study of six bitter melon varieties grown in australia shows total phenolic content between 5.1 to 7.9 mg GaE/g, in water extract (tan et al. 2014) or even 10.7 ± 0.3 mg GaE/g if extracted with ethanol 80% (tan et al. 2014).the Romanian variety M.charantia 'Brâncuşi' fruits' total phenol content (27.64 ± 1.89 mg GaE/ml juice) and total flavonoid content (23.13 ± 1.33 mg QE/ml juice) are higher, as they are measured from the fresh juice (Busuioc et al. 2020).
the antioxidant activity of different species of Momordica has been intensively studied, and is described in several works (nagarani et al. 2014b;Chokki et al. 2020;Lin et al. 2020).
all tested samples were active on in vitro antioxidant tests (Figure S1).Stem and leaves were more active than fruits for the capture of free dppH radicals (iC 50 = 216.9± 11.91 µg/ml; iC 50 of ascorbic acid = 0.003706 ± 0.00035 µg/ml). in the aBtS assay, the antioxidant potential of young fruits (iC 50 = 213.9± 17.4 µg/ml) and ripe fruits (iC 50 = 223 ± 21.19 µg/ml; iC 50 of trolox = 1.5353 ± 0.13 µg/ml) was superior to stem and leaves (iC 50 = 351.9± 35.5 µg/ml).moreover, the fruits imported from india were not statistically superior to the 'Enaja' variety, when tested by the aBtS assay (iC 50 = 186.6 ± 8.344 µg/ml); in the dppH test, this sample proved to be at least active (iC 50 = 812.1 ± 31.7 µg/ml) (Figure S1).We must mention that the indian fruits were purchased online, from a drugstore, in dry and powdered form.thus, we do not know their stage of maturity or the method of drying.its lower antioxidant activity may be due to the oxidation that occurs during the drying or storage of the fruits (Horax et al. 2006;Lin et al. 2020).
a study of Busuioc et al. ( 2020) evaluated the antioxidant activity of bitter melon juice obtained from a Romanian variety, M.charantia 'Brâncuşi' .the results of the dppH assay revealed a 65% reduction in the dppH radical when mixing 100 µl juice with 100 µl of a 100 µg/ml dppH solution (Busuioc et al. 2020).
Statistical correlations have been studied between total phenol content and antioxidant activity determined by dppH and aBtS assays (see table S4).total flavonoid content was shown to provide the highest association with dppH assay in our study (pearson correlation coefficient r = 0.8806, r 2 = 0.7754).on the other side, pearson's correlation coefficient (r = 0.5781) with p < 0.05 showed a significant correlation between total phenol content and dppH.However, there was no significant correlation between tpC, ttC, tFC, and aBtS assay in the present study.Further studies are required to reveal the complex interrelationships of the different bioactive compounds involved in the antioxidant effect of bitter melon.overall, based on the previous publications in which bitter melon grown in Romania was examined (pop et al. 2015;popescu et al. 2016;Busuioc et al. 2020) and based on our results, we can say that this plant, even when grown in Romania, is a valuable source of polyphenols and antioxidants.

Experimental
See supplementary material.