UPLC-ESI-TQD-MS/MS Identification and Antioxidant, Anti-Inflammatory, Anti-Diabetic, Anti-Obesity and Anticancer Properties of Polyphenolic Compounds of Hawthorn Seeds

Hawthorn seeds are a by-product of fruit processing and due to the scale of processing of this raw material, they can be an important source of bioactive compounds. This work is the first report on the phenolic composition of hawthorn seeds and their antioxidant, anti-inflammatory, antidiabetic, antiobesity and anticancer activities. In the isolated phenolic fraction of six seed species, 23 phenolic compounds were identified using the UPLC-ESI-TQD-MS/MS method, the key ones of which included the B-type procyanidin dimer. The seeds of the tested species showed high antioxidant activity (mainly by scavenging O2•− and OH• radicals), anti-inflammatory (mainly through LOX inhibition), anti-diabetic, anti-obesity and anti-cancer, with the highest activity against colon cancer cells (Dld-1 line), showing no activity against healthy colon epithelial cells (CCD841CoN). This activity was significantly dependent on the analyzed hawthorn species and, according to PCA analysis, on the content of flavan-3-ols. These discoveries provided the theoretical basis for the possibility of industrial use of hawthorn seeds. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s11130-024-01197-4.


Introduction
Hawthorn (Crataegus L.) belongs to the rose family (Rosaceae) and the apple subfamily (Maliodeae).The estimated number of hawthorn species in the world ranges from 20 to 185.This plant is widely distributed in North America, Europe, Central and East Asia [1].Currently, the use of hawthorn as a natural health product has been recognized by the Pharmacopoeia.The medicinal raw material is the inflorescence (Crataegi folium cum florae) and fruit (Crataegi fructus) collected at full ripeness and dried [1,2].Hawthorn is considered one of the most valuable plant raw materials used in the treatment of cardiovascular diseases, and the beneficial health-promoting effects of this plant have been attributed to phenolic compounds [3][4][5].The fruits are dominated by oligomeric procyanidins and their glycosides, while the leaves and flowers are dominated by phenolic acids and flavonols.
However, compared to the morphological parts of the hawthorn bush discussed above, incomparably less research has been carried out on the chemical composition and possible health-promoting activity of hawthorn seeds.In Asia alone, the annual production of hawthorn exceeds 2.1 million tons [6].A by-product of medicinal and food processing of fruit are seeds, which, due to the scale of processing of this raw material, become available in huge quantities.Therefore, their full phytochemical characterization seems necessary to fully exploit their possible health-promoting potential.
Therefore, the aim of the study was to investigate the content of bioactive ingredients in the isolated phenolic fraction of the seeds of six species of hawthorn and their impact on the antioxidant (scavenging of ABTS, O 2 •− , OH • radicals, cupric reducing antioxidant capacity and chelation of iron ions), anti-inflammatory (LOX, COX-2, XO, trypsin inhibition), anti-diabetic (α-amylase), anti-obesity (lipase), anticancer against ten human cancer cell lines (Mcf-7, U87mg, U251mg, AGS, Ht-29, Caco-2, Ls180, Dld-1, Sk-mel-28, Jurakt) and cytotoxic to a healthy colonic epithelial cell line (CCD841CoN).Moreover, for better understanding of the data, Pearson correlation and PCA test were performed.The obtained results constitute the first comprehensive analysis of the polyphenol profile and health-promoting properties of hawthorn seeds, so they can be a good reference for the use of the bioactive substances they contain in the medical, cosmetic and food industries.

Materials and Methods
The material and methods section are presented as Supplementary Material.

Phenolic Profile
As mentioned, the proven clinical medicinal activity of hawthorn flowers and fruits has been attributed to the content of phenolic compounds.Therefore, the first step in the analysis of hawthorn seeds was to assess the phenolic profile.Depending on the species, the content of phenolic compounds ranged from 46.8 (C.laevigata) to 71.3 (C.macrocarpa) mg/g (Fig. 1, Table 1).23 compounds were identified in the phenol profile, of which 16 belonged to the flavan-3-ols group, 4 to flavonols and 3 compounds to the phenolic acids group.The quantitative profile of these groups of compounds was as follows: 84.6 -89.0%(flavan-3-ols) > 4.1 -10.8% (phenolic acids) > 3.9 -7.2% (flavonols).The content of individual phenolic compounds in the seeds of 6 hawthorn species is given in Table S2.The most numerous group of phenolic compounds in hawthorn seeds were flavan-3-ols, such as polymeric procyanidins.Their amount ranged from 40.4 (C.monogyna; C. laevigata) to 60.3 (C.macrocarpa) mg/g.Among the identified flavan-3-ols, the highest concentrations were: procyanidin dimer type-B (27.1-37.1%)> procyanidin trimer type-C (26.1-29.2%)> (+)catechin (19.2-28.1%).Another group of phenolic compounds identified in hawthorn seeds are phenolic acids.Their amount ranged from 2.1 (C.rhipidophylla) to 7.7 (C.macrocarpa) mg/g.Among the identified phenolic acids, the highest concentrations were: 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid and coumarylquinic acid, in the following proportions: So far, the phenolic profile of hawthorn seeds has been analyzed in only two studies.Salmanian et al. [7] and Niu et al. [8] identified three compounds in C. elbursensis seeds, such as caffeic acid, chlorogenic acid and gallic acid, with a total concentration 41.2 times lower than in this study.These differences may be due to the extraction method used in this work.As a result of extraction into the solid phase, the obtained preparation was cleaned of ballast substances, which resulted in an increase in the phenolic fraction.In our studies, phenolic acids accounted for only 4.1 -10.8% of the total identified phenols, and their content between species differed statistically significantly (p < 0.05).Nevertheless, this group of compounds dominates the phenolic composition of hawthorn flowers and leaves, accounting for 53 and 41% of all phenols, respectively [9].Żurek et al. concluded that the dominant compound in these parts of the plant is 3-O-caffeoylquinic acid [10].In turn, the dominant compounds in hawthorn fruits are flavan-3-ols, which constitute nearly 55% of their phenolic composition, and the largest amounts include: procyanidin dimer, procyanidin trimer and (+) catechin [9].Their total amount in hawthorn fruits is 1.3 times higher than in the tested seeds.Overall, the procyanidin fraction of hawthorn fruit is of great interest as important ingredients in nutrition with high biological activity, including antioxidant, antibacterial, antiviral, anticancer, anti-inflammatory, neuro-and cardioprotective activities [11].Han et al. [12] demonstrated a beneficial effect of procyanidin extract from hawthorn fruit on the intestinal microflora and the production of shortchain fatty acids in the colon.It is therefore worth noting that hawthorn seeds, a by-product of hawthorn fruit processing, are an important source of procyanidins, which, once isolated, can be used to develop preparations with targeted health-promoting effects.

Antioxidant Activity
Values for the ABTS and CUPRAC methods ranged from 5.8 to 8.2 mmol TE/g and from 9.6 to 12.9 mmol TE/g, respectively.O 2 •− and OH • radical scavenging activities ranged from 65.4 to 135.0 μg/mL and from 71.4 to 175.1 μg/mL, respectively (Table 2).In turn, for the ChP method the values ranged from 70.0 to 452.3 μg/mL.The antioxidant activity of the analyzed species varied and showed significant differences (p < 0.05).The highest antioxidant activity was found for the seeds of C. macrocarpa (ABTS, CURPAC method), C. laevigata x rhipidophylla x monogyna (O 2 •− and OH • ) and C. monogyna (ChP).The antioxidant activity of hawthorn seeds has not been assessed so far by any of the above methods.The available literature only contains data expressing the antioxidant activity of three parts of hawthorn fruit using the DPPH and FRAP test, in the order peel > seed > pulp [7,13].In turn, compared to other morphological parts of hawthorn, in relation to the present results, the antioxidant activity of the fruit was lower by 28.3 times (for the ABTS test) [14], 4.1 times (ChP) [15], 3.0 times (O 2 •− ) and 1.2 times higher (OH • ) [16].These significant differences in antioxidant activity can be attributed to the method of obtaining preparations for analysis.The cited works assessed mainly the so-called crude extracts, most often ethanol.However, in this study, the preparations were obtained as a result of SPE extraction, which allowed the removal of ballast substances, which resulted in an increased content of phenols and antioxidant activity.The relationship between antioxidant activity and the content of phenolic compounds was also confirmed by Pearson's correlation.A significant correlation was observed between the total content of phenols and flavan-3-ols and the ABTS test (flavan-3-ols r > 0.794; total phenols r > 0.781) and the CUPRAC test (flavan-3-ols r > 0.930; total phenols r > 0.938) and between flavonols and OH • scavenging (r > -0.889) (Fig. 2A).Similar observations apply to hawthorn fruits, for which a strong correlation with procyanidins was demonstrated, indicating that procyanidin B2 and (-)epicatechin are the most effective antioxidant compounds in hawthorn [17,18].
So far, it has only been reported that polysaccharides isolated from C. azarolus seeds have a threefold lower ability to inhibit α-amylase (IC 50 , 3.01 mg/mL) than pulp polysaccharides [19].There are no reports in the literature about the lipase and trypsin-inhibiting effects of hawthorn extracts.In this study, the activity of α-amylase inhibition was shown to be correlated with polyphenolic compounds, more precisely with flavonols (r > 0.805).In studies conducted on rats, the activity in the treatment of hyperglycemia was also attributed to hawthorn fruit flavonoids, mainly quercetin and hyperoside [20,21].The authors showed that extracts rich in these ingredients lower blood glucose levels, increase insulin release and inhibit the increase in postprandial glucose levels.However, weak correlations were noted for lipase and trypsin (r < 0.217).Therefore, the lack of relationship between the inhibition of the activity of these enzymes and the content of polyphenols may indicate that this activity may be influenced by other groups of compounds.Previously, Wojdyło et al. showed that terpenoids and alkaloids are strong lipase and trypsin inhibitors [22].

Anti-Inflammatory Activity
The highest LOX and COX-2 inhibition activity was demonstrated for the species C. macrocarpa.The obtained values ranged from 285.5 to 375.7 MIU/g and 2.1 -6.1 MIU/g, respectively (Table 2).In the case of XO, the inhibitory effect was much lower compared to COX-2 and LOX, and ranged from 135.7-149.6IU/g.The highest activity was demonstrated for the species C. laevigata x rhipidophylla x monogyna.Generally, no significant differences between species were found in the LOX test.The demonstrated antiinflammatory activity (for the COX-2 and XO tests) can be attributed to polyphenolic compounds, mainly the content of flavan-3-ols (Pearson correlation COX-2 vs. flavan-3-ols, r > 0.730; XO vs. flavan-3-ols, r > 0.658) and total phenols (COX-2 vs. total phenols, r > 0.686; XO vs. total phenols, r > 0.968).The relationship between anti-inflammatory activity and phenolic content has been reported previously.Cui et al. [23] and Wyspiańska et al. [24] examined the anti-inflammatory effects of hawthorn fruit and bark.They determined that the anti-inflammatory activity of these raw materials is correlated with the content of procyanidins, in particular epicatechin and procyanidin B. Also, Elango and Devaraj [25] for procyanidins extracted from hawthorn fruit showed the ability to alleviate the pro-inflammatory immune response in rats by significantly reducing the level of proinflammatory mediators (IL-1β, IL-6 i TNF-α).In general, the anti-inflammatory activity of procyanidins has been confirmed in numerous studies conducted on both animal and human cellular models of inflammation [26].

Cell Viability
In terms of the cytotoxic properties of hawthorn seeds towards the analyzed cancer lines, the assessed activity can be arranged in the following order: Dld-1 > Caco-2 > Ht-29 > Ls18 0 > U87 mg > AGS > M cf-7 > J urakt > U 251 mg > Sk-mel-28 (Table 2).T he hig hest anticancer ac tiv ity was demo nst rat ed against colorectal cancer cells.The obtained IC 50 values ranged from 94.6 to 200.9 (Dld-1) and 105.9 -212.4 (Ht-29) μg/mL.With respect to these cell lines, the highest activity was demonstrated by seeds of the species C. laevigata x rhipidophylla x monogyna and C. macrocarpa, and the lowest by C. monogyna and C. rhipidophylla.In turn, the lowest activity was found against malignant cancer cell lines, such as Sk-mel-28 and U251mg, which are characterized by high therapeutic resistance.This is the first report on the anticancer activity of hawthorn seeds.
Other authors examining hawthorn fruits also reported the highest activity against colorectal cancer cells.It has been proven that hawthorn polysaccharides inhibit the proliferation of colon cancer cells (HCT116) by arresting the cell cycle in the G2/M and S phase and inducing apoptosis as a result of activation of P38 kinase [27].In our previous work, we showed that among the fruits, leaves and flowers of hawthorn, the fruits have the strongest anticancer activity [9].Their activity against glioblastoma cells (U87mg) depended on the concentration of flavan-3-ols and caused cell apoptosis by cutting PARP1, inhibiting the activity of FAK and Akt kinases, indicating the suppression of the proliferative and invasive potential.Also in this study, the anticancer activity was strongly dependent on the content of flavan-3-ols.Between this group of compounds and all analyzed cancer cell lines, the Pearson correlation coefficient was r > -0.695.It is worth emphasizing that hawthorn seeds did not show any cytotoxicity towards normal colonic epithelial cells (> 750 µg/mL), which indicates the safety of use of the obtained preparations.

PCA
The PCA plot presented 79.30% of the total variance in the data, with PC1 and PC2 explaining 62.12% and 17.18% of the total variance, respectively (Fig. 2B).The first group contained seeds of the species C. laevigata x rhipidophylla x monogyna, C. macrocarpa and C. subsphaericea, which were characterized by a high content of flavonols, phenolic acids, flavan-3-ols (procyanidin dimer type-B, (+) catechin, procyanidin trimer), as well as high antioxidant activity, expressed by the ABTS, CUPRAC, O 2 •− and OH • method, XO and COX-2 inhibition, anti-diabetic and anticancer activity.The second group included the species C. monogyna, which was characterized by high anti-obesity and metal ion chelating activity.The third group consisted of seeds of C. rhipidophylla and C. laevigata species, which were characterized by anti-inflammatory activity (LOX and trypsin inhibition).It can be assumed that the health-promoting activity of seeds belonging to scaled groups 2 and 3 depended on ingredients other than phenolic compounds.Overall, the PCA analysis confirmed our previous conclusions, showing that the hawthorn species had a significant impact on the content of phenolic compounds and healthpromoting activity.

Conclusion
This study provided missing data on the phenolic composition of hawthorn seeds and their broad health-promoting activities in vitro.The evaluated hawthorn seeds were particularly rich in flavan-3-ols, which constituted 84.6 -89.0% of the total quantitative phenolic composition, where the dominant compounds were procyanidin dimer type-B and procyanidin trimer type-C.The highest concentration of these compounds was recorded for the species C. macrocarpa.High antioxidant activity was observed for the tested seeds through various mechanisms of action, anti-diabetic, anti-obesity and anti-inflammatory activity, including the highest activity towards LOX.Cytotoxic activity towards cancer cells, in particular colon cells, was also found, with no toxicity towards healthy colon epithelial cells.The highest values of the mentioned health-promoting properties were found in seeds of the species C. macrocarpa and C. laevigata x rhipidophylla x monogyna, whose high activity correlated with the content of flavan-3-ols.
Due to current consumer demands, it is necessary to analyze and promote unconventional raw materials and their ingredients with high potential for industrial use.The obtained results should particularly arouse the interest of the medical, cosmetic and food industries in the use of bioactive ingredients and health-promoting properties of hawthorn seeds in the development of attractive products with potential health benefits.

Table 1
Individual phenolic compounds identified by UPLC-ESI-TQD-MS/MS in seeds hawthorn

Table 2
Antioxidant, anti-inflammatory, anti-obesity, anti-diabetes, anticancer activity of seeds of six hawthorn species Results are expressed as mean and SD.Significant differences between species were assessed by Duncan's test (p < 0.05)