C. pentagyna extraction yield, quantitative evaluation of phenolic profile, and antioxidant power.

The chemical components of the fruit, leaf, and root of C. pentagyna were fractionated using ether petroleum, 80% aqueous methanol, and ethanol as extraction solvents. The yield of extracts varied between 2.4% and 9.6% for methanolic extracts, between 2.1% and 8.3% for ethanolic extracts, and between 0.9% and 3.6% for petroleum ether extracts. Due to the hydroxyl (-OH) and methoxy (-OCH3) groups in their molecular structures [32], phenolic compounds possess the ability to scavenge free radicals. The phenolic content of hawthorn varies by cultivar, species, geographical location, harvest time, method of chemical determination of phytochemicals, and extraction preparation conditions [33]. There are few studies on the phytochemical content of C. pentagyna compounds. In addition, there have been no reports of hawthorn root. In this study, the phytochemical content (phenol, flavonoid, phenolic acid, and anthocyanin) and antioxidant capacity (DPPH scavenging) of hydro-methanolic and hydro-ethanolic extracts of fruit, leaf, and root were evaluated and compared (Table 1). The results indicated that hydro-methanolic extracts contain more phytochemicals and have more antioxidant capacity than ethanol extracts. Fruit extract contained the highest concentrations of phenolic (210.22 ± 0.44 mg GAE/g DE), total flavonoid (79.93 ± 0.54 mg QE/g DE), total phenolic acid (194.64 ± 0.32 mg CAE/g DE), and total anthocyanin (85.37 ± 0.13 mg cyanidin 3-glucoside/100 g FW) among the tested extracts, followed by leaf and root extracts. The total content of phenol and flavonoid in methanolic extracts of fruit from various regions ranged from 69.12 to 186.72 mg GAE/g and 1.6 to 85.31 mg QE/g dry weight plant, respectively, as reported by other researchers [15, 32, 34,35,36]. In a different study, the TPC and TFC concentrations in methanolic leaf extracts were 206.94 GAE/g and 57.08 mg (+)-catechin/g, respectively [37, 38]. There was a strong correlation between TPC and DPPH reduction. The phenolic and flavonoid content of fruit, leaf, and root extracts increases their antioxidant activity. The fruit extract with the highest phenolic and flavonoid content exhibited the highest DPPH radical scavenging capacity (IC50 = 15.43 ± 0.65 g/mL), followed by leaf and root extracts (IC50 = 34.67 ± 0.14 g/mL and 60.72 ± 0.32 g/mL, respectively). Moreover, the fruit and leaf extracts exhibited potent activity comparable to the positive control butylate hydroxytoluene (BHT) (IC50 = 49.02 ± 0.2 g/mL), whereas the root extract was less active. Antioxidant activity of fruit and leaf extracts of C. pentaegyna from different regions have been already investigated. For the methanolic fraction of fruit, the IC50 values for DPPH radical scavenging activity range from 17.48 to 341.29 g/mL [32, 34, 36, 39]. Moreover, the DPPH inhibition of C. pentagyna leaf was quantified as 5708 g/mL in hydroacetonic extract and 2.34 M TE/g (micromoles of Trolox equivalents) in ethanolic extract, respectively [18, 37, 40].

Table 1 Phytochemical screen and antioxidant activity of fruit, leaf, and root of C. pentagyna (TP: total phenol; TF: total flavonoid; TPA: total phenolic acid; TAC: total anthocyanin)Phenolic characterization using HPLC-PDA and LC/ESI-MS/MS

Using HPLC-ESI-MS/MS in negative ionization mode, the phytochemical profile of hydro-methanolic extracts of the fruit, leaf, and root of C. pentagyna tentatively were identified. Fig. S1 A-C depict the HPLC-ESI-MS/MS and HPLC-PDA (280 nm, 330 nm, and 360 nm) fingerprints (Supplementary materials). LC-ESI-MS/MS chromatograms show total ion chromatograms of compounds (TIC). The extracted ion chromatograms (XIC) from total ion chromatograms were processed by MZmine analysis software package which can separate all chromatograms and compounds. Figure 1A-C illustrates the instances of extracted ion chromatograms (XIC). According to the ESI-MS/MS fragmentation pattern, molecular weights, matching mass adducts ([M-H]−, [2M]−, [2 M-H]−, [M-2 H]−,[M-2 H + Na]− and [M-2 H + K]−) and published data, we identified 49, 42, and 33 phenolic compounds from the fruit, leaf, and root, respectively (Table 2) and (Supplementary materials: Table S1 and Fig S3-S5). Other hawthorn species (Crataegus spp.) in which these compounds have been previously identified are listed in Table 2 to compare with obtained results in our study. Phenolic compounds of C. pentagyna have been identified in a limited number of studies. Salmanian et al. [16]. determined the concentrations of gallic acid, caffeic acid, and chlorogenic acid in pulp and seed extracts of C. pentagyna from Iran. In Austrian C. pentagyna leaves, Prinz et al. [17]. isolated and identified hyperoside, rutin, isoquercitrin, sexangularetin-3-O-glucoside, isoorientin, isoorientin-2-O-rhamnoside, isovitexin, orientin, orientin-2-O-rhamnoside, vitexin, and vitexin-2-O-rhamnoside as flavones. In a separate study, Pavlovic et al. [18]. determined phenolics (p-coumaric acid, chlorogenic acid, caffeic acid, ferulic acid, isoquercetin, quercetin, rutin, (-)-epicatechin, kaempferol 3-O-glucoside, hyperoside, apigenin, cyanidin 3-O-glucoside, luteolin, procyanidins B1 and B2) during harvesting period in C. pentagyna fruits, flowers and leaves from Serbia. In addition, Alirezalu et al. [15]. quantified chlorogenic acid, vitexin, hyperoside, rutin, quercetin, and isoquercetin in the Iranian C. pentagyna fruit extract. Moreover, a number of 39 compounds (flavonoid aglycones, flavonoid O- and C-glycosides, organic and phenolic acids and proanthocyanidins) were identified in Romania C. pentagyna leaf, flower and fruit ethyl acetate extracts [19].

Fig. 1

Extracted ion chromatograms (XIC) and corresponding mass adducts in the hydro-methanolic extracts of C. pentagyna. (A) chromatogram XIC of sinapic acid and mass adducts, m/z 223; (B) XIC of Kaempferol-3-O-rutinoside and mass adducts, m/z 285; and (C) XIC of naringenin-7-O-glucoside and mass adducts, m/z 433

Table 2 Phenolic constituents identified in the hydro-methanolic extracts of the fruit, leaf, and root of C. pentagyna using HPLC-ESI-MS/MS.

Below is an explanation of the HPLC-ESI-MS/MS identification of phenolics in fruit, leaf, and root, as well as the comparison of literature.

Flavonoids

Flavonoids containing two benzene rings and one oxygenated ring were the most abundant phenols found in hawthorn in this study. According to Table 2, the aerial parts and roots of C. pentagyna contain 63 phenolic compounds. In mass spectrometry, all O-glycosides, including glucose or galactose (162 Daltons), rhamnose (146 Daltons), pentose—xylose or arabinose (132 Daltons), and disaccharide structures—rutinose or neohesperidose, lost their sugar moiety (308 Daltons). Due to cross-ring cleavages of sugar residues, C-glycosides exhibited fragments at m/z [M-H-18]−−, [M-H-60]−, [M-H-90]−, [M-H-120]−, [M-H-180]−, and [M-H-210]− for pentosyl residues and [M-H-74]− and [M-H-104]− for deoxyhexosyl residues [41].

Identification of luteolin derivatives

Compounds 50, 40, and 41 were identified as luteolin 7-O-glucoside, luteolin-7-O-glucuronide, and luteolin, respectively. Previous studies have identified compound 41 in C. microphylla leaves [42]; compound 50 from fruits, leaves, and flowers of C. microphylla [12, 17, 18, 42]; and compound 40 from leaves of C. macrocarpa [43].

Identification of orientin derivatives

Compounds 36 and 37 were characterized as orientin or isoorientin; and compounds 22 and and 23 as orientin or isoorientin glycoside derivatives (orientin-2″-O-rhamnoside and isoorientin-2″-O-rhamnoside. Previous studies have identified compound orientin from leaves and flowers of C. monogyna and C. pentagyna; compound isoorientin from flowers of C. monogyna and C. pentagyna; compound orientin-2″-O-rhamnoside from leaves and flowers of C. pentagyna; and compound isoorientin-2″-O-rhamnoside from flowers of C. pentagyna [12, 17].

Identification of quercetin derivatives

Compounds 48 was identified as quercetin aglycone. Compounds 31, 33 and 32 were identified as quercetin-O-glycoside derivatives (quercetin 3-O-glucoside (isoquercitrin), quercetin 4′-O-glucoside (spiraeoside) or quercetin 3-O-galactoside (hyperoside); and compounds 18, 21, 20 and 19 as quercetin-3-O-rutinoside, quercetin-3-O-rhamnoside, quercetin 7,4′-dimethyl ether-3-O-rutinoside and quercetin-3-O-(6″ galloyl) glucoside, respectively. Previous studies have identified compound 48 from fruits and leaves of C. pentagyna, C. monogyna, and C. oxyacantha, fruits of C. pinnatifida, C. germanica, C. cuneata, and C. brettschneideri, flowers and leaves of C. microphylla, leaves of C. scabrifolia and C. pinnatifid, and flowers of C. azarolus [18, 19, 42, 44, 45]; isoquercitrin from leaves of C. pentagyna, flowers of C. monogyna, C. macrocarpa, C. rhipidophylla, C. laevigata, and C. azarolus, and fruits of C. scabrifolia [12, 15, 18, 43, 46]; spiraeoside from fruits and flowers of C. monogyna and C. azarolus [1, 47,48,49]; hyperoside from fruits, leaves, and flowers of C. pentagyna, seeds and fruits of C. microphylla, C. macrocarpa, and C. oxyacantha, and leaves of C. pinnatifida [12, 15, 18, 42, 43, 50,51,52,53,54]; compound 18 from leaves of C. scabrifolia [55]; compound 21 from leaves and flowers of C. pentagyna [18]; and compound 20 from fruits of C. monogyna [56]; and compound 19 from leaves of C. monogyna [56].

Identification of kaempferol derivatives

Compounds 53, 50 and 38 were assigned as kaempferol aglycone, kaempferol-3-O-rutinoside and 8-methoxykaempferol (sexangularetin), respectively. Previous studies have identified compound 53 from fruits and flowers of C. pentagyna [18, 44]; compound 50 from fruits and leaves of C. pentagyna [18]; and compound 38 from flowers of C. maximowiczii [51].

Identification of apigenin derivatives

Compounds 56 and 42 were identified as apigenin aglycone and apigenin 8-C-glucoside (vitexin). Also, compounds 24, 25, 28 were detected as vitexin-O-rhamnoside derivatives (vitexin 2″-O-rhamnoside, isovitexin 2″-O-rhamnoside or vitexin-4′- O-rhamnoside); and compounds 29 and 26 as vitexin-O- glucoside (vitexin 4′-O-glucoside or vitexin-2″-O-glucoside), respectively. Previous studies have identified compound 56 from leaves of C. microphylla [42]; compound 42 from fruits, leaves, and flowers of C. pentagyna, C. monogyna, C. microphylla, and C. macrocarpa, and leaves C. pinnatifida [12, 15, 17, 42, 43, 51, 57]; vitexin-2″-O-rhamnoside from leaves and flowers of C. pentagyna, fruits, leaves, and flowers of C. monogyna and C. pinnatifida, leaves of C. microphylla, C. aronia, C. pseudoheterophylla, C. scabrifolia, and C. cuneata, flowers of C. macrocarpa, C. rhipidophylla, and C. laevigata, and fruits and leaves of C. pinnatifida [48, 58,59,60,61,62,63,64,65]; vitexin-4′-O-rhamnoside from leaves of C. oxyacantha and leaves and flowers of C. microphylla [42, 50, 64, 65]; vitexin-4′-O-glucoside from leaves of C. scabrifolia and C. cuneata, and fruits and leaves of C. pinnatifida [51, 64,65,66]; and vitexin-2″-O-glucoside from leaves of C. pinnatifida [58, 60].

Identification of eriodictyol derivatives

Compounds 54, 55 and 39 were assigned as eriodictyol aglycon, hesperetin and eriodictyol-7-glucuronide, respectively. Previous studies have identified compounds 54 and 55 from flowers of C. microphylla [42] and compound 39 from leaves and flowers of C. macrocarpa [43].

Identification of naringenin derivatives

Compounds 57, 27, 43 and 44 were suggested to be naringenin aglycon, naringenin 7-O-neohesperidoside (naringin), naringenin-6-C-glucoside and naringenin 7-O-glucoside, respectively. Previous studies have identified compound 57 from leaves of C. microphylla [42, 66]; compound 27 from leaves of C. oxyacantha [67]; and compounds 43 and 44 from leaves of C. monogyna and C. laevigata [68].

Identification of catechins, proanthocyanidins, and their derivatives

There are two major categories of procyanidins (A-type and B-type). (Epi)catechin units linked through C4 to C8 or C4 to C6 are called B-type procyanidins, while those with an additional bond (C2-O-C7) are called A-type. In the negative ion mode, there are three characteristic fragmentation routes, including retro Diels–Alder (RDA) [M-152-H]−, heterocyclic ring fission (HRF) [M-125-H]−, and quinine methide (QM) reaction (cleavage of the interflavan bond), with [M-289-H]− or [M-287-H]− as fragment ions of procyanidins [69]. Due to the degree of polymerization [70], procyanidins may exist as several isomers with the same molecular weight and mass spectrometry. In the present study, samples of C. pentagyna contained B-types and their deri