Evaluation of Apricot, Bilberry, and Elderberry Pomace Constituents and Their Potential To Enhance the Endothelial Nitric Oxide Synthase (eNOS) Activity.

Pomace, the press residue from different fruits accumulating as waste product in food industry, contains high amounts of secondary metabolites that could be utilized for health-related applications. This study aims at evaluating the potential of pomaces of apricot, bilberry, and elderberry to serve as a source for endothelial nitric oxide synthase (eNOS)-activating compounds. Five extracts obtained from the lyophilized pomace of apricot and elderberry with solvents of different polarity were found to enhance A23187-stimulated eNOS activity when tested at 50 μg/mL in an [14C]-l-arginine to [14C]-l-citrulline conversion assay in the human endothelium-derived cell line EA.hy926 (p < 0.05). The bioassay-guided fractionation of the extracts obtained with methanol/water (70:30) led to several active fractions from apricot pomace (p < 0.05) and elderberry pomace (p < 0.01). Liquid chromatography-mass spectrometry-based chemical analysis of the extracts and active fractions pointed mainly to triterpenoic acids as active compounds. One particular dihydroxytriterpenoic acid, characteristic for elderberry, was enriched as the main compound in the two most active fractions and might serve as a promising lead structure for further studies.

Introduction

Cardiovascular diseases (CVDs) are the leading cause of deaths worldwide, with ischemic heart disease and stroke together accounting for estimated 15.2 million deaths in 2016.[1] The number of deaths due to ischemic heart diseases has significantly increased in the last two centuries, and the death rates are particularly high in wealthy countries. Atherosclerosis has been evaluated as the first manifestation of cardiovascular diseases. A root cause of atherosclerosis is endothelial dysfunction, which is reversible by lifestyle changes, but in most cases leads to more severe conditions. Nitric oxide (NO) seems to play a key role in the maintenance of endothelial homeostasis and vascular health.[2,3] Vascular NO is mainly produced by the endothelial nitric oxide synthase (eNOS) in the endothelial cell layer. It, inter alia, mediates relaxation in vascular smooth muscle cells and influences their gene transcription/protein expression, inhibits platelet aggregation, and serves as a vascular antioxidant.

Although there are numerous causes and risk factors for CVDs, it is estimated that a high percentage is preventable by means of adaptations to lifestyle, i.e., by avoiding established risk factors such as smoking, excessive alcohol consumption, physical inactivity, and unhealthy diet. A fruit- and vegetable-rich diet is associated with a reduced risk for atherosclerosis,[4] hypertension,[5,6] and cardiovascular diseases[7] and indeed compounds from nutritional sources, such as quercetin,[8] (−)-epicatechin,[9] or ursolic acid,[10] have been shown to increase the eNOS activity in vascular endothelial cells. Pomace, the press residue from fruit juice, fruit nectar, or cider industry, still contains high amounts of fruit-derived constituents,[11,12] suggesting possible health-related applications. So far, some industrial uses for pomaces are already in place, such as pectin extraction, solid-phase fermentation, and animal feed. However, pomaces incur in tons during the fruit press seasons and confront juice producers with storage problems and disposal obligations; thus, further applications are of high interest. This is already under investigation for apple pomace,[13,14] but should also be considered for other pomaces.

In this study, we aimed to identify and characterize extracts, enriched fractions, or single compounds from apricot (Prunus armeniaca L.), bilberry (Vaccinium myrtillus L.), and elderberry (Sambucus nigra L.) pomace that are able to increase NO production in human endothelium-derived cells. To this end, we tested whether extracts obtained with solvents of different polarity enhance A23187-stimulated eNOS activation in EA.hy926 cells using the [14C]-l-arginine to [14C]-l-citrulline conversion assay (ACCA), in which [14C]-l-citrulline production serves as a surrogate marker of liberated NO. The chemical characterization of the extracts by liquid chromatography–mass spectrometry (LC–MS) identified several flavonoids, fatty acids, and triterpenoic acids that were previously shown to mediate beneficial effects on eNOS activation in vivo and/or in vitro. Bioassay-guided fractionation led to the conclusion that several di- and trihydroxylated triterpenoic acids are most likely responsible for the increase in A23187-stimulated eNOS activity observed for several fractions and extracts.

Results and Discussion

Pomace Extraction

Solvents of different polarity, namely, methanol/water (70:30) (MeW), ethylacetate (EtOAc), dichloromethane (DCM), and hexane (HEX), were used for extraction. Drug/extract ratios (DERs) are listed in Table . The MeW extraction resulted in the highest yields for all pomace samples, showing that polar constituents such as carbohydrates make up the largest part of all pomaces. This is particularly the case for apricot pomace, whose extractable matter is predominately composed of polar compounds, whereas the berry pomaces also contain significant amounts of substances that are preferentially extracted with DCM or HEX.

Table 1

Extraction Yields of Fruit Pomaces Using Pressurized Liquid Extraction (Dionex ASE 200) Expressed as Drug (=Pomace) Extract Ratios (DERs)

	DER
pomace	MeW	EtOAc	DCM	HEX
apricot	1.4:1	16.0:1	80.0:1	96.7:1
bilberry	8.7:1	12.2:1	11.3:1	13.1:1
elderberry	12.7:1	27.0:1	23.8:1	32.3:1

Potential of Pomace Extracts to Enhance eNOS Activation

Twelve extracts from three different fruit pomaces (Table ) were screened for their ability to enhance A23187-stimulated activation of eNOS in the immortalized human endothelium-derived cell line EA.hy926. For this, the [14C]-l-arginine to [14C]-l-citrulline conversion assay (ACCA) with ascorbic acid (100 μM) as positive control (PC) and solvent vehicle (dimethyl sulfoxide, DMSO) as negative control (NC) was applied. The assay was performed 24 h after cell treatment to allow the detection of influences on eNOS activity on the transcriptional, translational, or post-translational level.[15,16]

When tested at a concentration of 50 μg/mL, two apolar extracts (EtOAc, DCM) of apricot pomace significantly increased the eNOS activity compared to the negative control (p value 0.05, Figure A). None of the bilberry pomace extracts showed any effect on eNOS activity in EA.hy926 cells (Figure B), whereas three of four elderberry pomace extracts exhibited a significant increase (p value 0.05, Figure C). With a 1.5-fold increase over the negative control, the elderberry pomace MeW extract showed a higher effect than the DCM and HEX extracts. Cell viability was controlled by the WST-1 assay. The metabolic activity of EA.hy926 cells was impaired by the elderberry HEX extract (p value 0.01), but not by the MeW and DCM extracts (Figure ). Consequently, the elderberry pomace was considered as the best source for compounds able to enhance eNOS activation.

Figure 1

A23187-stimulated eNOS activity in EA.hy926 cells measured by the arginine–citrulline conversion assay (ACCA) after 24 h of treatment with 50 μg/mL MeW, EtOAc, DCM, and HEX extracts of apricot pomace (A), bilberry pomace (B), and elderberry pomace (C). NC, negative control (DMSO); PC, positive control (100 μM ascorbic acid); and one-tailed Mann–Whitney test (n = 3; mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 2

Metabolic activity of EA.hy926 cells measured by the WST-1 assay after 24 h of treatment with eNOS-activating extracts from elderberry pomace (=50 μg/mL MeW, DCM, and HEX extracts of elderberry pomace). NC, negative control (DMSO); PC, positive control (100 μM ursolic acid); one-tailed Mann–Whitney test (n = 3; mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001).

Identification of the Main Compounds in Pomace Extracts by HPLC–DAD–CAD and HPLC–ESI–MS Analysis

The obtained extracts were chemically characterized by high-performance liquid chromatography (HPLC)–diode array detector (DAD)–charged aerosol detector (CAD) and HPLC–electrospray ionization (ESI)–MS analyses, and the results are presented in Table . All MeW extracts consisted mainly of polar compounds that were not or only weakly retained on the C18 column material (Figure ), of which only citric acid (1) was identified by comparison with a reference (Figure S4). Several flavonoids and anthocyanins were elucidated in the berry pomace MeW extracts as less polar constituents. The most abundant ones were cyanidin-3-glucoside (7), cyanidin-3-sambubioside (2),[17] and rutin (17)[18] (Figure S5) from elderberry and hyperoside (19)[19] (Figure S6), as well as numerous glycosides of cyanidin, delphinidin, petunidin, peonidin, and malvidin (3–16)[20] from bilberry.

Table 2

Molecular Weight (MW) and Proposed Structures of the Main Compounds in the Fruit Pomace Extracts

compound number	retention time in gradient program 1 (GP1) or 2 (GP2)	Da	[M – H2O + H]+	[M + H]+ or M+	[M + Na]+	[M – H]−	proposed structure	references
1	4.2–4.4 (GP1)	192.0			215.0	190.9	citric acida
2	14.6–15.3 (GP1)	581.1		581.1			cyanidin-3-O-sambubiosideb	(17)
3		465.0		465.0			delphinidin-3-O-galactosideb	(20)
4		465.0		465.0			delphinidin-3-O-glucosideb	(20)
5		435.0		435.0			delphinidin-3-O-arabinosideb	(20)
6		449.0		449.0			cyanidin-3-O-galactosideb	(20)
7		449.0		449.0			cyanidin-3-O-glucosideb	(17, 20)
8		449.0		449.0			petunidin-3-O-arabinosideb	(20)
9		479.3		479.0			petundin-3-O-galactosideb	(20)
10		479.3		479.0			petundin-3-O-glucosideb	(20)
11		493.0		493.0			malvidin-3-O-galactosideb	(20)
12		493.0		493.0			malvidin-3-O-glucosideb	(20)
13		463.0		463.0			peonidin-3-O-galactosideb	(20)
14		463.0		463.0			peonidin-3-O-glucosideb	(20)
15		463.0		463.0			malvidin-3-O-arabinosideb	(20)
16		419.0		419.0			cyanidin-3-O-arabinosideb	(20)
17	17.0 (GP1)	610.0		611.0		609.0	rutina	(31)
18	17.1 (GP1)	536.1	519.0			535.1	coumaroyl iridoid isomerb	(32)
19	17.4 (GP1)	464.0		465.0		463.0	hyperosidea	(19, 32)
20	9.2 (GP2)	180.0		181.0			unknown 1
21	15.2–15.3 (GP2)	488.3		489.3		487.3	trihydroxytriterpenoic acid 1b	(33)
22	15.8–15.9 (GP2)	488.3		489.3		487.3	trihydroxytriterpenoic acid 2b	(33)
23	17.4 (GP2)	488.3	471.2	489.1	511.3	487.4	trihydroxytriterpenoic acid 3	(33)
24	17.7 (GP2)	488.3	471.2	489.3	511.3	487.3	trihydroxytriterpenoic acid 4	(33)
25	19.3 (GP2)	742.4			765.3	741.4	PI (16:0/9:0(COOH)
26	22.1–22.3 (GP2)	472.1	455.2		495.3	471.3	dihydroxytriterpenoic acid 1b	(33)
27	24.9–25.0 (GP2)	472.3		473.3		471.3	dihydroxytriterpenoic acid 2b	(33)
28	24.9–25.0 (GP2)	634.3		635.3		633.4	coumaroyl-dihydroxytriterpenoic acid 1b	(33)
29	25.3–25.8 (GP2)	472.1	455.2		495.3	471.6	dihydroxytriterpenoic acid 3b	(33)
30	25.3–25.8 (GP2)	472.1	455.2		495.3	471.6	dihydroxytriterpenoic acid 4b	(33)
31	26.1–26.2 (GP2)	472.3		473.3		471.3	dihydroxytriterpenoic acid 5b	(33)
32	26.1–26.2 (GP2)	634.3		635.3		633.4	coumaroyl-dihydroxytriterpenoic acid 2b	(33)
33	26.4–26.5 (GP2)	470.3		471.3		469.3	oxohydroxytriterpenoic acid 1b	(33)
34	27.3–27.4 (GP2)	472.3		473.3		471.3	dihydroxytriterpenoic acid 6b	(33)
35	28.3–28.4 (GP2)	472.3		473.3		471.3	dihydroxytriterpenoic acid 7b	(33)
36	33.0–33.2 (GP2)	470.3		471.3		469.3	oxohydroxytriterpenoic acid 2b	(33)
37	33.1 (GP2)	606.3	589.3		629.3		unknown 2
38	36.0 (GP2)	514.3			537.2	513.3	acetoxyhydroxytriterpenoic acid
39	36.8 (GP2)	518.3	501.3		541.3	517.5	unknown 3
40	38.2 (GP2)	518.3	501.3		541.3	517.5	unknown 4
41	38.2–38.4 (GP2)	456.3		457.3		455.3	betulinic acidb	(13, 33)
42	40.1–40.3 (GP2)	456.3		457.3		455.3	oleanolic acida	(13, 33)
43	40.4–40.7 (GP2)	456.3		457.3		455.3	ursolic acida	(13, 33)
44	40.6 (GP2)	665.3		666.3	688.4	664.3	PC (16:0/9:0(COOH))
45	48.5–48.6 (GP2)	438.3		439.3		483.5	ursadienoic or oleanadienoic acidb	(34)
46	50.0 (GP2)	498.3		499.3		497.3	acetoxytriterpenoic acid
47	55.7 (GP1)	280.1		281.2		279.1	linoleic acid (18:2)a
48	51.4–51.7 (GP2)	256.2		257.2		255.2	palmitic acid (16:0)a
49	51.4–51.7 (GP2)	282.2		283.2		281.2	oleic acid (18:1)b	(35)
50	56.0 (GP2)	460.2	443.2		483.3	459.2	unknown 5
51	56.1 (GP2)	358.2	341.2		381.2		unknown 6
52	57.8–58.0 (GP2)	462.3	445.3			461.3	unknown 7
53	59.7 (GP2)	284.4				283.4	stearic acid (C18:0)	(36)

Identification confirmed by standard addition experiments.

Proposed structure according to literature references.

Figure 3

HPLC profiles of the MeW extracts from apricot, elderberry, and bilberry pomace. Column: Hypersil BDS C18 (250 × 4.0 mm; 5 μm); mobile phase A: H2O, pH 2.8 (HCOOH); mobile phase B: ACN (HCOOH); flow rate: 1 mL/min; column oven: 17 °C; injected volume: 5 μL; elution gradient: 1–95% B in 60 min; detection: CAD.

Triterpenoic acids and fatty acids were already detected in the MeW extracts (Figure ) but became the dominant compound classes in all apolar EtOAc, DCM, and HEX extracts (Figure S1).

In all three pomaces, oleanolic acid (42) and ursolic acid (43) were the main triterpenoic acids (Figure S7). Apricot pomace displayed the highest diversity of triterpenoic acid species, with numerous trihydroxy- (21, 22), dihydroxy- (27, 31, 34, and 35), and coumaroyldihydroxytriterpenoic acids (28, 32) along with betulinic acid (41) and an acetoxytriterpenoic acid (46) being present particularly in the EtOAc and DCM extract (Figure S1). Another dihydroxytriterpenoic acid (26) was specific for elderberry, whereas both elderberry and bilberry contained relatively low amounts of two oxohydroxytriterpenoic acids (33, 36) and a compound tentatively identified as oleanadienoic or ursadienoic acid (45).

Regarding fatty acids, linoleic acid (47) was abundant in the elderberry and bilberry MeW extracts (Figure ), whereas palmitic acid (48) (Figure S7), oleic acid (49), and stearic acid (53) were found in different concentrations in all apolar extracts (Figure S1).

Particularly in case of the HEX extracts, which generally showed the lowest peak numbers of all extracts, it is likely that additional compounds are present that are too apolar to be detected by the applied LC–MS method.

Bioactivity-Guided Fractionation of the Pomace MeW Extracts

To identify the active components that increase eNOS activity, the methanol/water extracts were chosen for bioassay-guided fractionation for the following reasons: (a) the elderberry MeW extract was an effective enhancer of eNOS activity, (b) extractable polar bulk compounds, such as carbohydrates and organic acids, may mask bioactivities of lower concentrated compounds in the other MeW extracts, (c) MeW yielded the highest amounts of extract for all investigated pomaces, (d) methanol comprises comparable dissolving properties to ethanol, a solvent that obliges minor restrictions in food and pharmaceutical industry, and (e) the MeW extracts contained the highest variety of compounds, ranging from very polar to apolar, with most of the compounds detected in the apolar extracts (EtOAc, DCM, HEX) being also present in the MeW extract, just less abundant.

The fractionation of the MeW extracts from apricot, bilberry, and elderberry pomace by column chromatography on styrene–divinylbenzene resulted in 24, 36, and 30 cumulative fractions, which were screened (n = 1) for their potential to increase eNOS activity (Figure S2). Fractions that showed positive effects in the initial ACCA screening were retested twice to reaffirm their bioactivity (Figure ).

Figure 4

A23187-stimulated eNOS activity in EA.hy926 cells measured by the arginine–citrulline conversion assay (ACCA) after 24 h of treatment (10 μg/mL) with cumulative fractions obtained from the fractionation of apricot pomace MeW extract (A), bilberry pomace MeW extract (B), and elderberry pomace MeW extract (C). The data from the screening of all fractions are shown in Figure S2. NC, negative control (DMSO); PC, positive control (100 μM ascorbic acid); one-tailed Mann–Whitney test (n = 3; mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001).

The fractions obtained from the bilberry MeW extract showed no significant effect on eNOS (Figure B) and were thus not further investigated. Fractionation of the apricot pomace MeW extract yielded fractions able to moderately enhance eNOS activation (Figure A). Phytochemical analysis revealed that one of them (fraction 193–202) was enriched in triterpenoic acids (21, 22, 23, 24, 27, 28, 31, 32, 34, 35, 41, 42, and 43), particularly the more polar ones already detected in the active EtOAc and DCM extracts (Figure S3). The other, more apolar two fractions (fraction 203–218, fraction 219–238) were less complex (Figure S3). One major compound in both fractions was an unknown acetoxytriterpenoic acid (46). Furthermore, an acetoxyhydroxytriterpenoic acid (38) and two fatty acids were identified in fraction 203–218, whereas fraction 219–238 contained an oxidized lipid (44)—besides some unidentified compounds, which were present in both fractions.

From elderberry pomace MeW extract, a significant increase in eNOS activity was affirmed for seven cumulative fractions (Figure C), of which, three most active ones were analyzed in detail (Figure ). The LC–MS analyses of fractions 211–215 and 216–221 revealed that the most abundant constituent of both is a dihydroxytriterpenoic acid (26), probably 20β-hydroxyursolic acid according to literature,[21,22] accompanied by an unknown compound (51). The third active fraction from elderberry pomace (242–255) showed only peaks of very low intensity, such as those of ursolic and oleanolic acid (42, 43), and probably contains compounds that escape detection by the applied LC–MS method.

Figure 5

Chromatographic profiles of eNOS-activating fractions from elderberry pomace MeW extract. Column: Hypersil BDS C18 (250 × 4.0 mm; 5 μm); mobile phase A: H2O; pH 2.8 (HCOOH); mobile phase B: ACN (HCOOH); flow rate: 1 mL/min; column oven: 25 °C; injected volume: 5 μL; gradient program 2 (GP2): 35–95% B in 60 min; and detection: CAD.

Although the main compounds in these active fractions were elucidated as triterpenoic acids, which are generally known for apoptotic or cytotoxic effects,[23] the metabolic activity of EA.hy926 cells was actually increased by these fractions (Figure ).

Figure 6

Metabolic activity of EA.hy926 cells measured by the WST-1 assay after 24 h of treatment (10 μg/mL) with eNOS-activating fractions from elderberry pomace MeW extract. NC, negative control (DMSO); PC, positive control (100 μM ursolic acid); and one-tailed Mann–Whitney test (n = 3; mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001).

Because only fatty acids and triterpenoic acids were identified as major compounds in the active MeW fractions and apolar extracts, their potential to mediate beneficial effects on eNOS activity is discussed.

Considering the identified fatty acids, an increase in basal eNOS activity in EA.hy926 cells was reported upon treatment with palmitic acid (48),[24] whereas Couloubaly et al.[24] reported a decrease in basal eNOS activity and eNOS-serine 1177 phosphorylation in EA.hy926 cells after stimulation with 100 μM linoleic acid (47)–albumin complex for 24 h. A contribution of fatty acids to the activity of some fractions and extracts can thus not be excluded.

Triterpenoic acids, which made up the largest fraction of the extractable apolar compounds, comprise various biological activities.[23] Focusing on eNOS activation, ursolic acid, for instance, was shown to mediate the acute NO-dependent vasorelaxant effects of Lepechinia caulescens (Ortega) Epling[25] and the cardiovascular protective effects of the Chinese drug Danshen (i.e., Salvia miltiorrhiza Bunge).[10] Increased eNOS promotor activity, eNOS mRNA and protein expression, and an increased phosphorylation at eNOS serine 1177 were shown to contribute to the enhanced NO production mediated by ursolic acid.[10,26] Rios et al.[27] predicted the affinities of plant-derived triterpenoic acids to two binding pockets of eNOS by computational docking experiments.

However, triterpenoic acids are also known for their inhibition of cell proliferation and their cytotoxicity. He and Liu[28] reported half maximum effective concentrations (EC50) for cytotoxicity of nine triterpenoic acids from apple peel in different cancer cell lines between 18.2 and 332 μM. Yamaguchi et al.[29] showed that 20 μM ursolic acid inhibited the growth of tumorigenic HM-SFME-1 cells to 10% and of nontumorigenic SFME-1 cells to 40% relative to the control. Further, it has been shown that ursolic acid concentrations above 5 μM significantly reduce the cell viability of coronary artery endothelial cells.[26]

The fractionation of the apricot and elderberry pomace MeW extracts hinted toward a higher eNOS-activating potential of fractions rich in di- and trihydroxylated triterpenoic acids compared to that containing predominantly oleanolic and ursolic acid. This result confirms the previous data obtained for apple pomace.[16] The activity of fractions obtained previously from apple pomace and now from apricot pomace MeW extract seems to be mediated by a mixture of several triterpenoic acids that are not necessarily active as single substance. In contrast, the two most active fractions from elderberry pomace MeW extract were of low complexity. The same main compound—a dihydroxytriterpenoic acid (26) that is probably 20β-hydroxyursolic acid—was detected in both fractions. These fractions clearly did not reduce the cell metabolic activity. The isolation, structure elucidation, and investigation of the mechanism of action of this compound would certainly be of interest but was not achieved in this study due to the low amounts of the relevant fractions.

As outlined above, several abundant compounds of the investigated fruit pomace extracts can be associated with beneficial effects on the activity of eNOS based on in vivo and/or in vitro studies. The eNOS in the endothelial cell layer is mainly responsible for the release of vascular NO, which protects the cardiovascular system in several ways: it regulates the vascular tone via relaxation of the vascular smooth muscle cells, has antithrombotic effects, serves as a vascular antioxidant, and attenuates the adhesion of leukocytes to the vascular endothelium. Although it is well established and broadly communicated that a fruit- and vegetable-rich diet is associated with a reduced risk for cardiovascular diseases,[4−7] changing their lifestyle proves to be very difficult to many people. Food supplements that are highly enriched in fruit-derived compounds that enhance eNOS in vivo could be one option in the prevention of cardiovascular diseases. Our results indicate that fruit pomaces might serve as a useful resource for the production of such food supplements, but further studies, particularly in vivo data, are required on the way to develop such a product.

Conclusions

In this study, constituents extracted from apricot, bilberry, and elderberry pomaces were analyzed by LC–MS and evaluated on their potential to beneficially influence the activity of eNOS. The majority of the extractable matter is made up of very polar compounds, but only the elderberry methanol/water (70:30) (MeW) extracts enhanced A23187-stimulated eNOS activity in the human endothelium-derived cell line EA.hy926 at 50 μg/mL. In addition, four of the more apolar extracts from the apricot and elderberry pomaces were active in the ACCA. The bioassay-guided fractionation of the apricot and elderberry pomace MeW extracts indicates that di- and trihydroxylated triterpenoic acids contribute strongly to the observed activity, whereas the major constituents oleanolic and ursolic acid are less or not effective. One particular dihydroxytriterpenoic acid from elderberry pomace, probably 20β-hydroxyursolic acid, might serve as a promising lead structure for further studies. It was highly enriched in fractions that enhanced A23187-stimulated eNOS activity at 10 μg/mL, but that did not reduce the cell metabolic activity, although triterpenoic acids are generally associated with cytotoxic effects. Provided that our in vitro data can be translated to positive effects in vivo, apricot and particularly elderberry pomaces might become valuable resources for health-related applications, such as the production of food supplements that reduce the risk for cardiovascular diseases.

Methods

Reagents

Methanol (MeOH) and hexane (HEX), both AnalaR Normapur, dichloromethane (DCM) and ethyl acetate (EtOAc), both GPR Rectapur, and acetonitrile (ACN) HiPerSolv Chromanorm for HPLC–CAD–MS analysis were obtained from VWR (Vienna, Austria). Formic acid (purity >98.0%) and conc. ammonia (24%) were purchased from Gatt-Koller (Absam, Austria). Water, for extraction and phytochemical analysis, was deionized and distilled. Betulinic acid (purity 95%), corosolic acid (purity 90%), maslinic acid (purity 89%), oleanolic acid (purity 94%) and ursolic acid (purity 94%), cyanidin-3-O-sambubioside (purity 99%), cyanidin-3-O-glucoside (purity 88%), delphinidin-3-O-glucoside (purity 87%), malvidin-3-O-glucoside (purity 99%), peonidin-3-O-glucoside (purity 94%), and petunidin-3-O-glucoside (purity 99%) were provided from Phytolab (Vestenbergsgreuth, Germany), hyperoside (purity 99%), and rutin (purity 99%) from Extrasynthèse (Lyon, France), and palmitic acid (purity 99%) and linoleic acid (purity 99%) from Sigma-Aldrich (St. Louis, MO).

Sample Collection and Preparation

Stone-free apricot pomace, from Obsthof Reisinger (Spitz/Donau, Austria), as well as elderberry pomace and bilberry pomace, both from RAUCH Fruchtsäfte (Rankweil, Austria), was immediately frozen (−20 °C) after pressing in the season 2011 and lyophilized for 48 h (Zirbus Va Co 5–11) before further processing. The apricot pomace was crushed using pistil and mortar, bilberry pomace was milled (RETSCH mill, sieve 0.5), and elderberry pomace was excluded from comminution treatment to avoid the release of cyanogenic glycosides due to the high stone content.[30] Addition of 20% diatomaceous earth to the homogeneous pomace powders avoided clotting during the pressurized liquid extraction process.

Extraction

Pressurized liquid extraction of homogenized pomace samples was carried out using an ASE 200 accelerated solvent extraction system (Dionex, Vienna, Austria) with methanol/water (70:30) (MeW), ethylacetate (EtOAc), dichloromethane (DCM), and hexane (HEX) as solvents. For each solvent, 8–10 g pomace or pomace/diatomaceous earth-mixture was filled into an 11 mL extraction cell and extracted in three cycles using the following conditions: preheat time: 5 min; heat time: 5 min; temperature: 40 °C; static extraction: 5 min; flush volume: 0.6%, purge time: 60 s; pressure: 1500 psi. The obtained extracts were dried by solvent evaporation and subsequent lyophilization, if necessary.

Fractionation of the MeW Extracts

Pomaces were extracted with MeW under conditions stated above but using 22 mL extraction cells filled with 15–17 g pomace or pomace/diatomaceous earth-mixture. The obtained pomace MeW extracts were dissolved in EtOH 96% (v/v)/water (50:50) and ultracentrifuged. The supernatant was pipetted in portions onto a styrene–divinylbenzene matrix (DIAION-HP 20), which previously has been washed with EtOH 96% (v/v) and conditioned with EtOH 96% (v/v)/H2O (10:90).

Fractions of 200 mL were eluted with a series of EtOH 96% (v/v)/H2O mixtures of decreasing polarity, in the case of elderberry and bilberry pomace, followed by MeOH and EtOAc. The fractions showing similar thin-layer chromatography (TLC) fingerprints were combined to cumulative fractions. Detailed information is given in Chapter 3 (Supporting Information).

[14C]-l-Arginine to [14C]-l-Citrulline Conversion Assay

The [14C]-l-arginine to [14C]-l-citrulline conversion assay (ACCA) was performed as described previously.[15,16] In brief, 5 × 105 EA.hy926 cells/well were seeded in 6-well plates and cultivated to confluence for 3–4 days. Before stimulation, 100 U/mL of bovine liver catalase (Sigma-Aldrich) was added to the culture media. Catalase prevents the accumulation of H2O2, which originates from reactions of the phenolic plant compounds with cell culture media components and may lead to false-positive results. For the assay, stock solutions of the extracts or fractions in DMSO were freshly diluted into the culture medium. Final DMSO concentrations did not exceed 0.1%. Negative control cells were treated with identical volumes of pure DMSO. Twenty-four hours after stimulation with either DMSO, as a negative control, or the indicated concentrations of the respective extracts and fractions, the medium was removed. The cells were washed and equilibrated for 15 min with 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethyl sulfonic acid (HEPES) buffer (10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethyl sulfonic acid (HEPES), 150 mM NaCl, 5 mM KCl from Carl Roth, 2 mM MgSO4, 10 mM glucose, 1.5 mM CaCl2·2H2O from Sigma-Aldrich, pH 7.4). Subsequently, 0.32 μM [14C]-l-arginine (346 mCi/mmol, New England Nuclear, Boston, MA) and 1 μM calcium ionophore (A23187, Alexis Biochemicals, Paisly, U.K.) were added. After 15 min of [14C]-l-citrulline production, the reaction was stopped by washing the cells with ice-cold phosphate-buffered saline (PBS). The cells were lysed with ice-cold ethanol 96% (v/v) and subsequently extracted with water. The supernatant was dried under vacuum (SPD 1010 Speed Vac, Thermo Savant). The obtained cell extract was redissolved in MeOH/H2O (1/1) and separation of [14C]-l-arginine and [14C]-l-citrulline was conducted via TLC (Polygram SIL N-HR, 20 × 20 cm, Machery-Nagel, Düren, Germany) using methanol/ammonia conc./chloroform/water (9:4:1:2) as mobile phase. Dried plates were autoradiographed by a phosphoimager (BAS-1800II, Fujifilm, Germany) and AIDA software (raytest, Langenzersdorf, Austria) was used for densitometric analysis. l-Ascorbic acid (100 μM, Sigma-Aldrich) was used as positive control.

Evaluation of Cell Viability

Viable, metabolically active cells convert the tetrazolium salt WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitro-phenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) to a water-soluble formazan derivative with high absorption rates at 440 nm. WST-1 was used according to the protocol given by the supplier (Roche Diagnostics, IN). The evaluation was performed 24 h after stimulation with the indicated concentrations of the respective extracts or fractions with correction for the self-absorption of the samples. Ursolic acid was used as a positive control.

Statistical Analysis

Raw data from the ACCA and WST-1 assay were normalized to the negative control (NC). Normalized data were incorporated in GraphPad Prism version 4.0 (La Jolla, CA). Significant differences with the NC were calculated using one-tailed Mann–Whitney Test. Shown are the mean values ± SD of three independent experiments.

Phytochemical Analysis

Sample Preparation

MeW extracts were dissolved in MeW to a concentration of 5.0 mg/mL, EtOAc, DCM, and HEX pomace extracts in MeOH/DMSO (80:20) in a concentration of 8.0 mg/mL. The samples were sonicated for 10 min, centrifuged at 13.4 × 103 rpm, and the supernatant taken for analysis. The precipitate was dried using a centrifugal evaporator (Genevac, Ipswich, U.K.) and weighed for calculating the percentage of dissolved extract, representing the phytochemically characterized part of the extract (Table S1).

Fractions were dissolved in MeOH/DMSO (80:20) at concentrations of 8 mg/mL.

HPLC–DAD–CAD and HPLC–MS Analysis

Analyses were carried out on an UltiMate 3000 RSLC-series system (Dionex/Thermo Fisher Scientific, Germering, Germany) coupled in parallel to a Corona ultra RS charged aerosol detector (CAD, Dionex/Thermo Fisher Scientific) and an HCT 3D quadrupole ion trap mass spectrometer equipped with an orthogonal ESI source (Bruker Daltonics, Bremen, Germany). The separation was performed on an Agilent Hypersil BDS C18, 4.0 × 250 mm, 5 μm column using water with pH 2.8 (formic acid) as mobile phase A and acetonitrile, modified with the same amount of formic acid, as mobile phase B. The HPLC flow rate was 1.0 mL/min. Gradient program 1 (GP1), used for MeW extracts, consisted of a linear increase from 1% B to 95% B in 60 min at 17 °C column oven temperature. Gradient program 2 (GP2), conducted for EtOAc, DCM, and HEX extracts, started with a concentration of 35% mobile phase B, which increased by 1% per min at 25 °C column oven temperature. The final mobile phase B concentration in both methods was 95%, which was held for 10 min to wash the column between the single runs, followed by 10 min of equilibration at starting conditions of the respective gradient program. As the primary objective was the phytochemical comparison of the different pomace extracts and fractions, the HPLC methods were not further optimized for particular pomace extracts or fractions.

After passing the DAD, the eluate flow was split 4:1 between the CAD and the MS, respectively. The CAD nebulizer temperature was 35 °C and the ESI ion source was operated as follows: capillary voltage: +3.5/–3.7 kV, nebulizer: 26 psi (N2), dry gas flow: 9 L/min (N2), and dry temperature: 340 °C. Positive and negative ions mode multistage mass spectra up to MS4 were obtained in automated data-dependent acquisition (DDA) mode using helium as collision gas, an isolation window of Δm/z = 4, and a fragmentation amplitude of 1.0 V.