Chemical Analysis of Dietary Constituents in Rosa roxburghii and Rosa sterilis Fruits.

Both Rosa roxburghii and R. sterilis, belonging to the Rosaceae, are endemic species in Guizhou Province, China. The fruits of these two species are mixed-used as functional food in the region. Aiming to elucidate the phytochemical characteristics of R. roxburghii and R. sterilis fruits, the essential oils and constituents in a methanol extract have been analyzed and compared by GC-MS and UFLC/Q-TOF-MS, respectively. As a result, a total of 135 volatile compounds were identified by GC-MS and 91 components were different between R. roxburghii and R. sterilis fruits; a total of 59 compounds in methanol extracts were identified by UFLC/Q-TOF-MS, including 13 organic acids, 12 flavonoids, 11 triterpenes, nine amino acids, five phenylpropanoid derivatives, four condensed tannins, two stilbenes, two benzaldehyde derivatives and one benzoic acid derivative; and nine characteristic compounds were found between R. roxburghii and R. sterilis fruits. This systematic study plays an important role for R. roxburghii and R. sterilis fruits in the product development.

1. Introduction

Rosa roxburghii (Figure 1), belonging to the Rosaceae family, originates from the karst areas of Guizhou Province, China and the effect of promoting digestion of its fruit was firstly recorded in “Ben-cao-gang-mu-shi-yi” in 1765 A.D. [1]. Modern pharmacological studies have proven that R. roxburghii fruit processes antioxidant, antimutagenic, antiatherogenic and antitumor effects, as well as genoprotective and radioprotective activities [2,3,4,5]. Due to the beneficial effects, a number of phytochemical studies have been performed on this species, and various phytochemicals, including flavonoids, organic acids, triterpenes, amino acids and essential oils have been found in R. roxburghii fruit [3,6,7,8,9,10]. Among them, the flavonoids and organic acids in R. roxburghii fruit have been widely studied. The total flavonoid content in R. roxburghii fruit was 5981–12,895 mg/100 g dry weight, which was approximately 120–360 folds that in citrus [11]. The total content of the six organic acids (malic acid, lactic acid, tartaric acid, citric acid, oxalic acid and succinic acid) in R. roxburghii fruit was over 40 mg/g fresh weight [5,12]. Moreover, ascorbic acid, a well-known organic acid, was the most prevalent compound with 4500–6800 mg/100 g dry weight in R. roxburghii fruit [13]. To our best knowledge, ascorbic acid is an essential nutrient related to the biosynthesis of collagen and certain hormones, and is a potential substance to reduce the risk of some diseases (e.g., cancer, cardiovascular and neurodegenerative diseases). In view of the high contents and bioactive properties of flavonoids and ascorbic acid, R. roxburghii fruit has increasing applications to produce juice, wine and the preserved fruit can be used as a dietary supplement in the health-related industries.

Figure 1

Morphology of R. roxburghii (A–C) and R. sterilis (D–F) fruits.

R. sterilis (Rosaceae, Figure 1) is a newly found species in Anshun, Guizhou Province, described by Shengde Shi in 1985 [14]. R. sterilis have a very close genetic relationship to R. roxburghii based on Random Amplified Polymorphic DNA (RAPD) markers [15]. Recently, R. sterilis fruit has been mixed with R. roxburghii fruit in the food industry due to the fact they come from the same producing region (mainly in Guizhou Province) and similar taste. It is well known that the constituents that are responsible for the flavor and the bioactivities have a great effect on the application of plant materials. Up to now, a few kinds of ingredients in R. sterilis fruit, namely essential oils, triterpenes, amino acids and trace elements have been identified [14,16,17,18]. However, it is still difficult to estimate whether R. sterilis fruit could be used as a substitute for R. roxburghii fruit in the food industry. Thus, it is necessary to elucidate the chemical profiles of R. roxburghii and R. Sterilis fruits before these two fruits are well developed.

In recent years, gas chromatography-mass spectrometer (GC-MS) and ultra-fast liquid chromatography/quadrupole time-of-flight mass spectrometry (UFLC/Q-TOF-MS) have become powerful technologies for chemical identification in complex extracts due to their high resolution and low detection limit [19,20,21,22]. Therefore, in this study, the essential oils and constituents in methanol extracts of R. roxburghii and R. sterilis fruits were identified and compared by GC-MS and UFLC/Q-TOF-MS to elucidate their chemical characteristics.

2. Results and Discussion

2.1. Chemical Analysis and Comparison of Essential Oils by GC-MS

It is well known that essential oils comprise an important part of fruits. In this study, a total of 135 compounds were identified in R. roxburghii and R. sterilis fruits by GC-MS. As shown in Table 1 and Figure 2, R. roxburghii and R. sterilis fruits differed in the composition of their essential oils, and only 45 components were shared between them. Interestingly, aliphatic compounds were the major constituents in essential oils of R. roxburghii and R. sterilis fruits. In R. roxburghii fruit, 89 compounds were found, representing 98.88% of the essential oil and the ten components with higher relative peak area were n-hexadecanoic acid (16.06%), octadecane (8.16%), 9,12,15-octadecatrien-1-ol (6.66%), nonacosane (6.44%), cholestane (5.24%), β-sitosterol (4.60%), stigmastane (4.44%), tetracosane (3.24%), 9,12-octadecadienoic acid (2.92%), and 2,2′-methylenebis[6-(1,1-dimethylethyl)-4-methyl]phenol (2.78%); 91 compounds represented 93.19% of the essential oil of R. sterilis fruit and the ten components with higher relative peak area were n-hexadecanoic acid (20.86%), 9,12,15-octadecatrienoic acid (9.06%), octadecane (5.70%), heptadecane (5.38%), nonacosane (5.07%), cholestane (3.93%), stigmastane (3.27%), triacontane (2.88%), 2,2′-methylenebis[6-(1,1-dimethylethyl)-4-methyl]phenol (2.51%) and β-sitosterol (2.36%). The essential oils of R. roxburghii and R. sterilis fruits have been reported previously [8,11,12,14,15,16]. The essential oil compositions varied highly when different extraction methods were used, such as hydrodistillation, supercritical CO2 extraction, solvent extraction and solid-phase microextraction. Liang et al. [23] compared the volatile compounds of R. roxburghii fruit extracted by hydrodistillation and solvent extraction, and found that hydrodistillation was the most effective approach for the extraction of long chain fatty acids, such as hexadecanoic acid and 9,12-octadecadienoic acid, which is accord with our results. Zhang et al. [16] separated and identified 41 volatile compounds from R. roxburghii fruit by solid-phase microextraction and GC-MS, and confirmed that limonene, ethyl caprylate, ethyl caproate, β-chamigrene and guaiene were the main constituents. A total of 57 volatile compounds from R. sterilis fruit have been reported by Jiang et al. [24] and 1,2,3,4-tetrahydro-1,1,6-trimethylnaphthalene, tetradecane, β-selinene, hexanoic acid and dihydro-β-ionol were the main constituents, which were obviously different from those (β-sitosterol, hentriacontane, octacosane, hexanoic acid and 11-(pentan-3-yl) henicosane) obtained by supercritical CO2 extraction [25]. Moreover, a higher relative intensity was found in the GC-MS chromatogram of R. sterilis fruit, indicating the content of essential oils in R. sterilis fruit was more than in R. roxburghii fruit using the same extraction method. This was consistent with the reports that extraction rates of essential oils were 1.8% and 0.8 for R. sterilis and R. roxburghii, respectively [23,25]. Among the 45 shared compounds, most of them have higher relative intensity and peak area in R. sterilis fruit than in R. roxburghii fruit, except for 5,6-dimethyldecane (17). In the present study, more constituents were separated and identified in the essential oils of R. roxburghii and R. sterilis fruits than in the previous studies using hydrodistillation. Ninety-one volatile compounds were different in these two species, which might explain their different smell and flavor.

Table 1

Volatile compounds identified in R. roxburghii and R. sterilis fruits by GC-MS (n = 3).

No.	Rt (min)	Compounds	Molecular Formula	Molecular Weight	Main Mass Fragments	Area% in RR Fruit	Area% in RS Fruit
1	3.12	3-Furaldehyde	C5H4O2	96	67, 39	-	0.01 ± 0.001
2	3.17	Furfural	C5H4O2	96	67, 39	0.67 ± 0.03	0.57 ± 0.02
3	3.34	3-Hexen-1-ol	C6H12O	100	83, 67, 41	-	0.53 ± 0.01
4	3.40	4-Methyl-octane	C9H20	128	112, 85, 71, 69, 43	0.83 ± 0.04	0.17 ± 0.01
5	3.41	Ethyl benzene	C8H10	106	91, 74, 51, 27	-	0.20 ± 0.02
6	3.48	o-Xylene	C8H10	106	91, 74, 51	0.3 ± 0.003	-
7	3.48	p-Xylene	C8H10	106	91, 74, 51	-	0.28 ± 0.02
8	3.67	Styrene	C8H8	104	78, 51, 27	1.53 ± 0.05	0.04 ± 0.003
9	4.27	5-Methyl-nonane	C10H22	142	112, 85, 65, 43	0.16 ± 0.003	-
10	4.94	2-Ethyl-1-hexanol	C8H18O	130	112, 83, 57, 29	0.23 ± 0.01	-
11	4.99	d-Limonene	C10H16	158	137, 121, 93, 68, 41	0.22 ± 0.01	0.17 ± 0.01
12	5.02	Benzyl alcohol	C7H8O	108	79, 51	-	0.02 ± 0.002
13	5.07	1,6-Dimethylhepta-1,3,5-triene	C9H14	122	107, 91, 65, 41	-	0.03 ± 0.002
14	5.16	3,7-Dimethyl-1,3,6-octatriene	C10H16	136	121, 93, 77, 57	-	0.05 ± 0.01
15	5.69	4,5-Diethyloctane	C12H26	170	141, 111, 84, 67, 43	0.63 ± 0.02	0.46 ± 0.02
16	5.94	1,2,4,5-Tetramethylbenzene	C10H14	134	119, 91, 65	-	0.01 ± 0.001
17	6.01	5,6-Dimethyldecane	C12H26	170	141, 113, 84, 67, 43	0.73 ± 0.02	0.18 ± 0.02
18	6.17	4-Ethyldecane	C12H26	170	140, 113, 85, 43	0.18 ± 0.01	-
19	6.24	2,6-Dimethyldecane	C12H26	170	140, 113, 71, 43	0.29 ± 0.01	-
20	6.68	Dodecane	C12H26	170	141, 112, 85, 57, 41	0.14 ± 0.01	-
21	7.05	2-Carene	C10H16	136	121, 93, 77	-	0.03 ± 0.003
22	7.22	2,6,10-Trimethyldodecane,	C15H32	212	183, 155, 127, 85, 57	-	0.02 ± 0.002
23	7.29	1,2,3,4-Tetrahydro-1,1,6-trimethylnaphthalene	C13H18	174	159, 131, 105, 71	-	0.07 ± 0.01
24	7.46	3-Methylnonadecane	C20H42	283	253, 169, 141, 113, 85, 57	0.12 ± 0.01	-
25	7.65	2,3-Dihydro-1,1,5,6-tetramethyl-1H-indene	C6H7BClNO3	187	159, 128, 91, 71	-	0.26 ± 0.02
26	7.66	Decane	C10H22	142	99, 71, 43, 27	0.03 ± 0.004	-
27	8.23	1,1,5-Trimethyl-1, 2-dihydronaphthalene	C13H16	172	157, 141, 115, 77	-	0.07 ± 0.01
28	8.28	Megastigma-4,6(Z),8(Z)-triene	C13H20	176	161, 133, 105, 77	-	0.04 ± 0.004
29	8.47	Dichloroacetic acid, tetradecyl ester	C16H30Cl2O2	325	196, 168, 139, 111, 83, 65, 43	0.07 ± 0.006	-
30	8.47	1-Tetradecene	C14H28	196	168, 140, 111, 83	-	0.04 ± 0.003
31	8.53	Tetradecane	C14H30	198	169, 141, 113, 85, 57	0.03 ± 0.003	-
32	8.74	Alloaromadendrene	C15H24	204	189, 161, 119, 91, 69	-	0.06 ± 0.004
33	8.99	3,8-Dimethyldecane	C12H26	170	141, 113, 85, 57	-	0.08 ± 0.01
34	9.04	9-Methylnonadecane	C20H42	282	267, 238, 168, 140, 113, 85	-	0.03 ± 0.004
35	9.20	2,6-Bis(1,1-dimethylethyl)-2,5-cyclohexadiene-1,4-dione	C14H20O2	220	177, 135, 91, 67	-	0.07 ± 0.01
36	9.51	2,4-Bis(1,1-dimethylethyl)phenol	C14H22O	206	191, 163, 115, 91, 57	0.13 ± 0.01	0.13 ± 0.02
37	9.60	1-Butyl-2-propylcyclopentane	C12H24	168	140, 111, 91, 69, 41	0.03 ± 0.004	-
38	9.65	4-Ethoxybenzoic acid ethyl ester	C11H14O3	194	149, 121, 93, 65	-	0.09 ± 0.01
39	9.87	Dodecanoic acid	C12H24O2	200	157, 129, 101, 73, 43	0.54 ± 0.02	2.26 ± 0.09
40	10.15	1-Tricosanol	C23H48O	340	322, 294, 154, 125, 97, 69, 43	0.08 ± 0.01	-
41	10.22	Hexadecane	C16H34	226	169, 141, 113, 85, 57	0.12 ± 0.02	-
42	11.26	2,6,10,14-Tetramethylpentadecane	C19H40	268	253, 183, 141, 113, 85, 57	-	0.20 ± 0.01
43	11.33	Heptadecane	C17H36	240	169, 141, 113, 85, 57	0.21 ± 0.01	5.38 ± 0.13
44	11.49	3,7,11-Trimethyl-1,6,10-dodecatrien-3-ol	C15H26O	222	204, 161, 136, 93, 69	0.24 ± 0.02	-
45	11.72	Hexacosane	C26H54	366	243, 197, 141, 113, 85, 57	-	0.09 ± 0.004
46	11.83	9-Octylheptadecane	C25H52	352	239, 197, 169, 141, 113, 85, 57	0.09 ± 0.01	-
47	11.86	Tetradecanoic acid	C14H28O2	228	185, 157, 129, 97, 73	0.44 ± 0.02	0.92 ± 0.02
48	12.08	Benzyl benzoate	C14H12O2	212	167, 105, 77, 51	-	0.27 ± 0.01
49	12.23	6-Tetradecanesulfonic acid butyl ester	C18H38O3S	334	196, 127, 91, 71, 52	-	0.08 ± 0.01
50	12.28	3,3,4-Trimethyl-4-p-tolylcyclopentanol	C15H22O	218	200, 163, 147, 119, 91	0.20 ± 0.01	-
51	12.50	2,6,10,14-Tetramethylhexadecane	C20H42	282	253, 183, 141, 113, 85	0.18 ± 0.01	-
52	13.06	2-Methylheptadecane	C18H38	254	239, 211, 141, 113, 85, 57	-	0.11 ± 0.01
53	13.25	Cyclohexadecane	C16H32	224	125, 55	-	0.16 ± 0.02
54	13.40	1,2-Benzenedicarboxylic acid bis(2-methylpropyl) ester	C16H22O4	278	223, 167, 149, 104, 76, 57	0.44 ± 0.03	0.28 ± 0.01
55	13.49	Cyclopentadecane	C15H30	210	182, 139, 111, 83, 55	0.07 ± 0.001	-
56	13.63	2-Methylhexacosane	C27H56	380	365, 337, 169, 141, 113, 85	-	0.10 ± 0.01
57	13.68	Hentriacontane	C31H64	436	169, 141, 113, 85, 57	-	0.14 ± 0.01
58	13.69	8-Heptadecene	C17H34	238	210, 140, 111, 83, 55	0.08 ± 0.001	-
59	14.21	2,3-Dimethylnonadecane	C21H44	297	253, 183, 155, 127, 99, 71	0.19 ± 0.01	-
60	14.53	Palmitoleic acid	C16H30O2	254	236, 192, 137, 111, 83	0.77 ± 0.02	-
61	14.57	Oxacycloheptadecan-2-one	C16H30O2	254	236, 194, 138, 111, 83	0.39 ± 0.01	-
62	14.87	n-Hexadecanoic acid a	C16H32O2	256	213, 185, 157, 129, 83, 60	16.06 ± 0.25	20.86 ± 0.31
63	15.64	1-Heptacosanol	C27H56O	396	378, 181, 153, 125, 97, 57	0.29 ± 0.02	-
64	16.06	3,7,11,15-Tetramethylhexadeca-1,3,6,10,14-pentaene	C20H32	272	229, 191, 119, 93, 69	0.65 ± 0.01	-
65	16.07	3,7,11-Trimethyl-2,6,10-dodecatrien-1-ol	C15H26O	222	191, 161, 137, 93, 69	-	0.70 ± 0.01
66	16.12	Trispiro[4.2.4.2.4.2.]heneicosane	C21H44	296	288, 231, 192, 163, 135, 97	0.29 ± 0.01	-
67	16.47	N-[4-bromo-n-butyl]-2-piperidinone	C9H16BrNO	234	205, 154, 97, 43	-	0.10 ± 0.01
68	16.73	2,2-Dimethyl-5-(3-methyloxiranyl)-cyclohexanone	C11H20O2	196	182, 153, 123, 95, 69, 41	0.11 ± 0.008	-
69	16.74	7-Bromomethylpentadec-7-ene	C16H31Br	302	223, 153, 125, 97, 69	-	0.15 ± 0.01
70	17.03	1-Nonadecene	C19H38	266	266, 210, 168, 126, 97	0.31 ± 0.01	0.10 ± 0.01
71	17.04	8-Hexadecene	C16H32	224	196, 153, 125, 97, 69	-	0.60 ± 0.02
72	17.21	Estra-1,3,5(10)-trien-17β-ol	C18H24O	256	185, 157, 129, 97, 73	-	0.11 ± 0.01
73	17.23	Nonadecyl pentafluoropropionate	C22H39F5O2	430	313, 266, 153, 125, 97, 57	0.03 ± 0.001	-
74	17.36	Heneicosane	C21H44	296	197, 169, 141, 113, 85	0.63 ± 0.02	0.51 ± 0.02
75	17.64	6-Octen-1-ol, 3,7-dimethyl acetate	C12H22O2	198	156, 123, 103, 81	2.33 ± 0.15	-
76	17.94	Trifluoroacetic acid pentadecyl ester	C18H31F3O2	336	306, 255, 182, 140, 111, 83	-	0.42 ± 0.02
77	18.09	9,12-Octadecadienoic acid a	C18H32O2	280	236, 150, 123, 95, 67	2.92 ± 0.09	0.59 ± 0.01
78	18.21	9,12,15-Octadecatrien-1-ol	C18H32O	264	236, 208, 108, 79	6.66 ± 0.14	-
79	18.40	9,12,15-Octadecatrienoic acid a	C18H30O2	278	222, 163, 135, 108, 79	-	9.06 ± 0.17
80	18.66	4-(4-Ethylcyclohexyl)-1-pentylcyclohexene	C19H34	262	220, 191, 164, 123, 81	0.42 ± 0.01	-
81	18.66	Methyl 6,9,12-hexadecatrienoate	C17H28O2	264	194, 175, 135, 107, 79	-	0.69 ± 0.02
82	18.80	9-Octadecenoic acid a	C18H34O2	282	264, 222, 165, 137, 111, 83	0.43 ± 0.01	0.14 ± 0.01
83	18.83	1-Eicosene	C20H40	280	252, 182, 153, 125, 97	0.53 ± 0.02	0.10 ± 0.01
84	18.84	tert-Hexadecanethiol	C16H34S	258	224, 165, 111, 57	-	0.74 ± 0.01
85	18.97	Bacchotricuneatin C	C20H22O5	342	245, 191, 145, 112, 71	-	0.14 ± 0.01
86	19.01	E-8-Methyl-7-dodecen-1-ol acetate	C15H28O2	240	197, 165, 126, 97, 69	0.08 ± 0.003	-
87	19.01	3-Methylheptadecane	C18H38	254	225, 169, 141, 113, 85, 57	-	0.12 ± 0.01
88	19.03	1-Chloro-octadecane	C18H37Cl	288	175, 147, 113, 85, 57	-	0.50 ± 0.01
89	19.15	2-Dodecen-1-yl(-)succinic anhydride	C16H26O3	266	237, 299, 181, 149, 123, 97, 69, 41	0.07 ± 0.001	0.10 ± 0.01
90	19.21	1-(1,5-Dimethylhexyl)-4-(4-methylpentyl)cyclohexane	C20H40	280	191, 166, 123, 97, 69	0.05 ± 0.001	0.27 ± 0.01
91	19.68	9-Tricosene	C23H46	322	294, 167, 139, 111, 83	0.95 ± 0.03	0.89 ± 0.02
92	19.83	13-Tetradecen-1-ol acetate	C16H30O2	254	194, 167, 139, 111, 83	0.13 ± 0.01	-
93	20.55	1-Tricosene	C23H46	322	196, 169, 139, 111, 83, 57	0.05 ± 0.001	-
94	20.92	1,7,11-Trimethyl-4-(1-methylethyl)cyclotetradecane	C20H40	280	236, 204, 165, 125, 97	0.10 ± 0.01	0.12 ± 0.01
95	21.03	1-Docosene	C23H46	308	223, 181, 139, 97, 57	0.33 ± 0.01	0.18 ± 0.01
96	21.34	Eicosyl pentafluoropropionate	C23H41F5O2	444	426, 280, 182, 153, 125, 97	0.09 ± 0.005	0.16 ± 0.01
97	21.66	Eicosane	C20H42	282	197, 169, 141, 113, 85	1.10 ± 0.04	2.28 ± 0.10
98	21.91	Docosane	C22H46	310	197, 169, 141, 113, 85	0.04 ± 0.003	0.37 ± 0.01
99	22.05	Nonadecane	C19H40	268	197, 169, 141, 113, 85	0.68 ± 0.01	-
100	22.06	Octatriacontyl trifluoroacetate	C40H77F3O2	646	181, 139, 97, 57	-	0.46 ± 0.01
101	22.61	Behenyl chloride	C22H45Cl	344	189, 169, 141, 113, 85	0.44 ± 0.01	0.05 ± 0.004
102	22.72	11,13-Dimethyl-12-tetradecen-1-ol acetate	C18H34O2	282	267, 208, 151, 123, 95, 69	0.10 ± 0.02	0.69 ± 0.02
103	22.72	15-Isobutyl-(13αH)-isocopalane	C24H44	332	276, 219, 191, 151, 123, 95	-	0.41 ± 0.01
104	24.21	Cyclotetracosane	C24H48	336	308, 167, 139, 111, 83	0.41 ± 0.01	0.49 ± 0.01
105	24.32	2,2'-Methylenebis[6-(1,1-dimethylethyl)-4-methyl]phenol	C23H32O2	340	284, 177, 149, 121, 91	2.78 ± 0.09	2.51 ± 0.07
106	24.59	Hexadecyloxirane	C18H36O	268	250, 211, 166, 138, 111, 82	0.71 ± 0.03	-
107	25.48	Dotriacontyl pentafluoropropionate	C35H65F5O2	612	594, 448, 181, 139, 97, 57	0.08 ± 0.002	0.13 ± 0.01
108	26.38	Tetratriacontane	C34H70	478	253, 225, 197, 169, 141, 113, 85	-	0.39 ± 0.02
109	26.66	12-Pentacosene	C25H50	350	350, 181, 153, 125, 97, 69	0.20 ± 0.01	-
110	27.32	Bis(2-ethylhexyl)phthalate	C24H38O4	390	279, 180, 149, 104, 57	1.60 ± 0.06	-
111	27.95	Eicosyl trifluoroacetate	C22H41F3O2	394	376, 325, 280, 153, 125, 97	0.06 ± 0.002	-
112	28.50	Heptacosyl trifluoroacetate	C29H55F3O2	492	474, 423, 378, 181, 139, 97	0.09 ± 0.01	-
113	28.58	Hexacosane	C26H54	366	169, 141, 113, 85, 57	1.11 ± 0.05	1.20 ± 0.06
114	29.31	2-Dodecen-1-yl(-)succinic anhydride	C16H26O3	266	209, 166, 137, 97, 69	1.65 ± 0.06	0.08 ± 0.002
115	29.53	Tricosane	C23H48	324	197, 169, 141, 113, 85	0.13 ± 0.01	0.53 ± 0.02
116	29.88	Pentacosane	C25H52	352	211, 169, 141, 113, 85, 57	-	1.56 ± 0.05
117	29.89	2,6,10,14,18-Pentamethyleicosane	C25H52	352	253, 183, 141, 113, 85	1.15 ± 0.04	-
118	30.64	2,6,10,14-Tetramethyl-7-(3-methylpent-4-enylidene)pentadecane	C25H48	348	264, 207, 167, 125, 97	0.56 ± 0.02	-
119	30.67	14-Nonacosane	C29H60	408	378, 181, 153, 125, 97	0.25 ± 0.01	0.10 ± 0.03
120	30.87	Octadecane	C18H38	254	169, 141, 113, 85, 57	8.16 ± 0.14	5.70 ± 0.09
121	31.05	1-Hexacosene	C26H52	364	209 ,181, 153, 125, 97	0.46 ± 0.01	0.54 ± 0.02
122	31.59	1-Bromo-11-iodoundecane	C11H22BrI	362	281, 233, 177, 135, 97	0.31 ± 0.01	0.27 ± 0.01
123	31.62	1-Methyl-4-(1-methylethyl)-3-[1-methyl-1-(4-methylpentyl)-5-methylheptyl]cyclohexene	C25H48	348	248, 193, 123, 69	0.24 ± 0.01	-
124	32.46	13-Methyl-Z-14-nonacosene	C30H60	420	405, 209, 181, 153, 125, 97	0.78 ± 0.02	-
125	32.98	(5α,14β)-Cholestane	C27H48	372	259, 218, 176, 149, 109	2.11 ± 0.03	1.66 ± 0.09
126	33.10	Tetracosane	C24H50	338	169, 141, 113, 85, 57	3.24 ± 0.05	1.00 ± 0.07
127	33.67	Cholestane	C27H48	372	262, 217, 149, 109, 81	5.24 ± 0.09	3.93 ± 0.10
128	34.33	(5α,13α)-d-Homoandrostane	C20H34	274	259, 217, 177, 149, 95, 55	1.25 ± 0.04	1.39 ± 0.06
129	35.31	Nonacosane	C29H60	408	197, 169, 141, 113, 85	6.44 ± 0.21	5.07 ± 0.19
130	35.94	Stigmastane	C29H52	400	290, 217, 189, 149, 109	4.44 ± 0.18	3.27 ± 0.14
131	37.45	1-Iodo-octadecane	C18H37I	380	253, 183, 141, 99, 57	2.16 ± 0.08	0.67 ± 0.03
132	37.46	Triacontane	C30H62	422	197, 169, 141, 113, 85, 57	-	2.88 ± 0.11
133	37.80	28-Nor-17α(H)-hopane	C29H50	398	383, 355, 218, 191, 137, 109	2.44 ± 0.09	-
134	43.31	β-Sitosterol	C29H50O	414	381, 329, 255, 213, 145, 81	4.60 ± 0.17	2.36 ± 0.09
135	43.70	(3β,24Z)-Stigmasta-5,24(28)-dien-3-ol	C29H48O	412	379, 314, 281, 229, 202	-	2.04 ± 0.10

a Compounds were confirmed by the reference standards. RR: R. roxburghii; RS: R. sterilis.

Figure 2

The representative total ion chromatograms of R. roxburghii (A) and R. sterilis (B) fruits obtained from GC-MS analysis.

Some main constituents in the essential oils have been reported for their pharmacological activities and nutritional values. For example, stigmastane and its derivatives possess anti-herpes virus and anti-inflammatory effects [26]. Meanwhile, 9,12,15-octadecatrienoic acid (linolenic acid), known as a vascular scavenger, has preventative effects against cardiovascular diseases, such as softening heart and brain blood vessels, promoting blood circulation and lowering blood pressure [27,28,29]. Therefore, elucidating the composition of the essential oils of R. roxburghii and R. sterilis fruits is useful for product development.

2.2. Chemical Analysis and Comparison of Multiple Constituents by UFLC/Q-TOF-MS/MS

2.2.1. Identification of Constituents

A total of 59 compounds were identified or tentatively characterized, including 13 organic acids (1, 5–6, 8–10, 12, 16, 19, 25, 28, 57 and 59), 12 flavonoids (18, 30–32, 34–36, 38, 44, 47, 48 and 49), 11 triterpenes (43, 45, 46, 50–56 and 58), nine amino acids (2–4, 7, 11, 13, 14, 17 and 20), five phenylpropanoid derivatives (26, 33, 39, 41 and 42), four condensed tannins (21, 24, 27 and 29), two stilbenes (37 and 40), two benzaldehyde derivatives (15 and 22) and one benzoic acid derivative (23). The detailed information is summarized in Table 2.

Table 2

Compounds identified in the methanol extracts of R. roxburghii and R. sterilis fruits.

No.	Rt (min)	Molecular Formula	[M + H]+	[M − H]−	Major Fragment Ions in Positive Mode	Major Fragment Ions in Negative Mode	Identification	Source
1	3.28	C3H6O3		89.02442 (0)		71.0233 [M − H − H2O]−	Lactic acid a	RR, RS
2	3.36	C3H7NO3	106.04983 (–0.4)	104.0534 (+0.1)	87.0324 [M + H − NH3]+		Serine a	RR, RS
3	3.4	C6H14N4O2	175.11826 (−4.0)	173.10527 (+5.0)	158.0918 [M + H − NH3]+, 130.0970 [M + H − NH3 − CO]+, 116.0724 [M + H − CH5N3]+		Arginine a	RR, RS
4	3.82	C5H9NO2	116.07018 (−3.7)	114.05579 (−2.3)	70.0677 [M + H − HCOOH]+		Proline a	RR, RS
5	3.92	C4H6O5		133.01458 (+2.5)		115.0047 [M − H − H2O]−	Malic acid a	RR, RS
6	3.99	C7H12O6	193.06974 (−4.8)	191.05529 (−4.3)		173.0462 [M − H − H2O]−, 127.0396 [M − H − H2O − HCOOH]−, 109.0294 [M − H − 2H2O − HCOOH]−	Quinic acid	RR, RS
7	4	C5H11NO2	118.08617 (−0.7)	116.07236 (+5.7)	72.0828 [M + H − HCOOH]+		Valine a	RR, RS
8	4.09	C6H8O6	177.03933 (−0.2)	175.02491 (+0.6)	129.0187, 111.0080, 95.0138	115.0043 [M − H − C2H4O2]−, 87.0103 [M − H − C2H4O2 − CO]−	Ascorbic acid a	RR, RS
9	5.18	C7H6O4	155.03362 (−1.7)	153.01985 (+3.4)	137.0253 [M + H − H2O]+	109.0.93 [M − H − CO2]−	Protocatechuic acid a	RR, RS
10	5.23	C6H8O7	193.03373 (−2.8)	191.02033 (+3.1)		155.0029 [M − H − 2H2O]−, 111.0079 [M − H − 2H2O − CO2]−	Citric acid a	RR, RS
11	5.65	C9H11NO3	182.08028 (−4.9)		147.0444 [M + H − H2O − NH3]+, 136.0761 [M + H − HCOOH]+, 119.0493 [M + H − C2H3NO2]+, 91.099 2[M + H − C2H3NO2 − H2O]+		Tyrosine a	RR, RS
12	5.65	C9H8O3	165.05427 (−2.1)	163.04076 (+4.2)	119.0467 [M + H − HCOOH]+, 91.0563 [M + H − HCOOH − H2O]+		p-Coumaric acid a	RR, RS
13	5.73	C6H13NO2	132.10173 (−1.3)	130.08736 (0)	86.0981 [M + H − HCOOH]+, 69.0722 [M + H − HCOOH − NH3]+		Isoleucine a	RR, RS
14	6.13	C6H13NO2	132.10198 (+0.6)	130.08852 (+9.0)	86.0982 [M + H − HCOOH]+, 69.0720 [M + H − HCOOH − NH3]+		Leucine a	RR, RS
15	6.99	C8H8O4	169.04931 (−1.3)	167.03526 (+1.7)	150.9672 [M+H − H2O]+, 95.0136	109.0268 [M − H − CO − HCOH]−	Vanillin a	RR
16	7.72	C7H6O5	171.02825 (−3.2)	169.0144 (+0.9)	139.0017, 111.0063	125.0238 [M − H − CO2]−	Gallic acid a	RR, RS
17	8.09	C9H11NO2	166.08574 (−3.1)	164.07209 (+2.4)	120.0809 [M + H − HCOOH]+, 103.0548 [M + H − HCOOH − NH3]+	147.0454 [M − H − NH3]−, 120.0444 [M − H − CO2]−	Phenylalanine a	RR, RS
18	9.27	C15H14O7	307.08106 (−0.6)	305.06686 (+0.6)		125.0234 [M − H − C8H8O4]−	Epigallocatechin	RS
19	10.07	C9H10O5		197.04591 (+1.8)		151.0430 [M − H − HCOOH]−, 125.0248 [M − H − CO2 − H2O]−	Syringic acid a	RR
20	10.14	C11H12N2O2	205.09718 (+0.1)	203.08256 (−0.2)	188.0691 [M + H − NH3]+, 170.0595 [M + H − NH3 − H2O]+, 146.0593, 118.0648	116.0507 [M − H − C3H7NO2]−	Tryptophan a	RR, RS
21	10.40	C30H26O12	579.14939 (−0.5)	577.13504 (−0.2)	427.1022 [M + H − C8H8O3]+, 409.0898 [M + H − C8H8O3 − H2O]+, 287.0554 [M + H − C15H16O6]+	451.1050, 425.0893 [M − H − C8H8O3]−, 407.0783 [M − H − C8H8O3 − H2O]−, 289.0729 [M − H − C15H12O6]−	Procyanidin B1	RR, RS
22	10.54	C9H10O4	183.06502 (−0.9)	181.05076 (+0.7)		163.0367 [M − H − H2O]−, 135.0449 [M − H − H2O − CO]−, 119.0495 [M − H − CH2O − CH4O]−	Syringaldehyde	RR
23	10.72	C13H16O8		299.0775 (+0.9)		137.0241 [M − H − glc]−, 93.0358 [M − H − glc − CO2]−	4-Hydroxybenzoic acid-4-O-glucopyranoside	RS
24	11.06	C30H26O12	579.14973 (0)	577.13567 (+0.9)	453.1169, 427.1037, 409.0923, 301.0721, 287.0554	289.0730 [M − H − C15H12O6]−	Procyanidin B2	RR, RS
25	11.73	C7H6O3	139.03867 (−2.2)	137.02506 (+4.7)	111.0440 [M + H − H2O]+, 95.0135 [M + H − CO2]+	93.0351 [M − H − CO2]−	4-Hydroxybenzoic acid a	RR, RS
26	12.39	C21H26O8	407.16816 (−4.6)		245.0449[M + H − glc]+		Erythro-guaiacylglycerol β-sinapyl ether or threo-guaiacylglycerol β-sinapyl ether	RR, RS
27	12.41	C30H26O11		561.14044 (+0.4)		407.0756 [M − H − C8H10O3]−, 289.0718 [M − H − C15H12O5]−, 273.07006 [M − H − C15H12O6]−	Fisetinidol-(4α,8)-catechin	RS
28	13.01	C9H8O4		179.03508 (+0.5)		135.0446 [M − H − CO2]−	Caffeic acid a	RR, RS
29	13.29	C30H26O12	579.14959 (−0.2)	577.13543 (+0.5)	439.1030, 427.1038 [M + H − C8H8O3]+, 409.0909 [M + H − C8H8O3 − H2O]+, 301.0738, 287.0554, 271.0620	451.1072, 425.0879 [M + H − C8H8O3]−, 407.0789 [M + H − C8H8O3 − H2O]−, 289.0716 [M − H − C15H12O6]−	Procyanidin B3	RR, RS
30	14.06	C27H30O16	611.16016 (−0.8)	609.14646 (+0.6)	303.0460 [M + H − rutinose]+	301.0333 [M − H − rui]−, 271.0238 [M − H − rui − CH2O]−	Rutin a	RR, RS
31	14.72	C21H20O12	465.10249 (−0.6)	463.08802 (−0.4)	303.0504 [M + H − glc]+	301.0347 [M − H − glc]−, 271.0260, 255.0292, 151.0027	Isoquercitrin a	RR, RS
32	15.18	C27H28O16	609.14377 (−2.0)	607.13045 (0)	303.0491	463.0839, 301.0352	Quercetin 3-O-[(X-O-3-hydroxy-3-methylglutaryl)-β-glucoside]	RR, RS
33	15.23	C22H26O8		417.15509 (−1.0)		181.0482 [M − H − C13H16O4]−,	Diasyringaresinol	RR
34	15.57	C20H18O11	435.09253 (+0.8)	433.07753 (−0.2)	303.0508 [M + H − C5H10O4]+	301.0358 [M − H − C5H10O4]−	Quercetin-3-O-d-xyloside	RR, RS
35	15.77	C21H20O11	449.10749 (−0.8)	447.09296 (−0.7)	303.0846 [M + H − C6H12O4]+, 151.0380, 123.0429	300.9984 [M − H − C6H12O4]−, 285.0406	Quercitrin a	RR, RS
36	15.94	C27H28O15	593.14886 (−2.1)	591.13566 (+0.2)	287.0543	529.1312, 489.1046, 447.0920, 285.0309	Kaempferol 3-O-[(X-O-3-hydroxy-3-methylglutaryl)-β-galactoside]	RR, RS
37	16.28	C20H22O8		389.12421 (0)		227.0712 [M − H − glc]−	Piceid	RR, RS
38	16.36	C27H28O15	593.14843 (−2.8)	591.13617 (+1.1)	287.0544	529.1236, 489.1044, 447.0936, 285.0407	Kaempferol 3-O-[(X-O-3-hydroxy-3-methylglutaryl)-β-glucoside]	RR, RS
39	16.83	C21H24O10		435.12995 (0.6)		273.0781[M − H − glc]−, 167.0349	Phloridzin	RR
40	17.21	C15H14O2	227.10655 (−0.4)		197.0650 [M + H − CH2O]+, 185.0997 [M + H − CH2O − H2O]+		3-Methoxy-5-hydroxy-stilbene	RR, RS
41	17.35	C21H26O8	407.16945 (−1.5)		389.1582 [M + H − H2O]+, 371.1472 [M + H − 2H2O]+, 245.1159 [M + H − glc]+, 215.1062, 199.1114		Erythro-guaiacylglycerol β-sinapyl ether or threo-guaiacylglycerol β-sinapyl ether	RR, RS
42	18.53	C20H22O6	359.14831 (−1.7)	357.13435 (0)	327.1216 [M − H − CH4O]−, 313.1057 [M − H − H2O − CO]−, 253.0856		Pinoresinol	RR, RS
43	18.68	C36H58O11		665.39109 (+0.7)		619.3935 [M − H − 2H2O]−, 485.3284 [M − H − glc − H2O]−, 441.3399 [M − H − glc − H2O − CO2]−, 357.2748	Polygalacic acid 3-O-β-d-glucopyranoside	RR, RS
44	18.9	C15H10O7	303.0497 (−0.7)	301.03548 (+0.4)	257.0431, 229.0497, 201.0540, 165.0182, 153.0181, 137.0230	178.9987, 151.0038, 121.0290	Quercetin a	RR, RS
45	19.38	C36H58O11		665.39114 (+0.8)		485.3272 [M − H − glc − H2O]−, 467.3211 [M − H − glc − 2H2O]−, 351.2653	19α -hydroxyasiatic acid-28-O-β-d-glucopyranoside	RR, RSS
46	20.39	C36H58O10		649.39552 (−0.3)		487.3427 [M − H − glc]−, 469.3319 [M − H − glc − H2O]−	Kajiichigoside F1	RR, RS
47	20.64	C15H10O6	287.05474 (−1.0)		165.0133, 153.0202		Kaempferol a	RR, RSS
48	22.10	C15H12O5	273.07551 (−0.9)		153.0181	151.003	Dihydroapigenin	RR, RS
49	22.95	C15H10O6	287.05465 (−1.3)	285.04066 (+0.7)	268.9789, 153.0188, 121.0251	229.0563, 187.0393, 169.0649, 143.0520	Luteolin a	RR, RS
50	26.48	C30H48O6		503.33793 (+0.2)		485.3320 [M − H − H2O]−, 439.3229 [M − H − HCOOH − H2O]−, 421.3141 [M − H − HCOOH − 2H2O]− 225.1623	1-Hydroxyeuscaphic acid	RR, RS
51	27.25	C30H46O5	487.34155 (−0.5)	485.32722 (−0.1)	451.3208 [M + H − 2H2O]+, 405.3170 [M + H − 2H2O − HCOOH]+, 223.1604, 199.1448, 187.1454	467.3176, 425.23124, 375.3047, 321.2564, 257.2382	2α,19α-Dihydroxy-3-oxo-urs-12-en-28-oic acid isomer	RR, RS
52	29.27	C30H48O5	489.35729 (−0.3)	487.34276 (−0.3)	407.3131 [M + H − 2H2O − HCOOH]+, 207.1733, 201.1624	469.3302 [M − H − H2O]− 443.3533 [M − H − CO2]−, 427.3205 [M − H − H2O − CO2]−, 371.2911	Euscaphic acid	RR, RSS
53	33.05	C30H46O5	487.34155 (−0.5)	485.32728 (+0.1)	451.3208 [M + H − 2H2O]+, 405.3170 [M + H − 2H2O − HCOOH]+, 223.1604, 199.1448, 187.1454	467.3186 [M − H − H2O]−, 441.3415 [M − H − CO2]−, 423.3280 [M − H − H2O − CO2]−, 393.3161	2α,19α-Dihydroxy-3-oxo-urs-12-en-28-oic acid	RR, RS
54	34.87	C30H48O4		471.34821 (+0.5)		453.3398 [M − H − H2O]− 423.3270, 407.3350, 377.2865	Pomolic acid or isomer	RR, RS
55	35.53	C30H48O4		471.348 (0)		453.3350 [M − H − H2O]−, 407.3315, 377.2888	Pomolic acid or isomer	RR, RS
56	35.56	C30H46O4		469.33187 (−1.0)		451.3237 [M − H − H2O]−, 407.3304 [M − H − H2O − CO2]−, 377.3222	2α,3β-Dihydroxylup-20(29)-en-28-oic acid	RR, RS
57	40.72	C18H30O2	279.2318 (−0.2)				9,12,15-Octadecatrienoic acid a	RS
58	42.58	C30H48O3	457.36755 (−0.2)	455.35307 (−0.6)		407.3335 [M − H − HCOOH]−	Ursolic acid a	RR, RS
59	42.68	C18H32O2	281.24735 (−0.6)	279.23322 (+1.0)		261.2124 [M − H − H2O]−	9,12-Octadecadienoic acid a	RR, RS

a Compound was confirmed by reference standard. RR: R. roxburghii; RS: R. sterilis.

Organic acids: Compared with the reference standards, peaks 1, 5, 8, 9, 10, 12, 16, 19, 25, 28, 57 and 59 were identified directly as lactic acid, malic acid, ascorbic acid, protocatechuic acid, citric acid, p-coumaric acid, gallic acid, syringic acid, p-hydroxybenzoic acid, caffeic acid, 9,12,15-octadecatrienoic acid and 9,12-octadecadienoic acid, respectively. The characteristic fragments were [M − H − H2O]−, [M − H − CO2]− and [M − H − HCOOH]− in negative ion mode. Compound 6 gave an [M − H]− at m/z 191.05529, corresponding to its elemental composition of C7H12O6. The high sensitive ions of m/z 173.0462 resulted from the loss of H2O, subsequently the loss of HCOOH yielded the ion m/z 127.0396. Therefore, compound 6 was tentatively identified as quinic acid [30,31].

Flavonoids: Peaks 30, 31, 35, 44, 47 and 49 were identified as rutin, isoquercitrin, quercitrin, quercetin, kaempferol and luteolin, respectively. The characteristic fragment of flavonoid glycoside was at m/z [aglycone]+ in positive ion mode and [aglycone]− by the loss of glycoside in negative ion mode. Referring to the compounds reported in R. roxburghii, peaks 18 (molecular formula: C15H14O7), 32 (molecular formula: C27H28O16), 36 (molecular formula: C27H28O15) and 38 (molecular formula: C27H28O15) were tentatively identified as epigallocatechin [6], quercetin 3-O-[(6-O-3-hydroxy-3-methylglutaryl)-β-galactoside] [32], kaempferol 3-O-[(X-O-3-hydroxy-3-methylglutaryl)-β-galactoside] [32] and kaempferol 3-O-[(X-O-3-hydroxy-3-methylglutaryl)-β-glucoside] [32], respectively. Peak 34 showed a deprotonated molecule ion at m/z 433.09253 in negative ion mode. The determination of fragment ion at m/z 301 confirmed the losses as C5H10O4, suggesting that the existence of xyloside group [9,33]. Peak 48 gave a protonated ion at m/z 273.07551 and the molecular formula was C15H12O5. According to MS data, fragment ion at m/z 153 was generated by retro Diels-Alder reaction in the C ring, which is the same as that happened in the standard compounds of quercetin, kaempferol and luteolin. Therefore, it was tentatively identified as dihydroapigenin [33].

Triterpenes: Peak 58 was identified as ursolic acid by comparison with the reference standard. Peaks 43 and 45 were a pair of isomers with a [M − H]− ion at m/z 665, corresponding to C36H58O11. The fragment ion at m/z 485, 180 Da less than molecular weight, hinted the presence of a glucose group. Then m/z 441 appeared behind m/z 485 in peak 43 of MS spectrum, indicating the presence of a carboxyl group. Considering the retention time and different fragments, the glucose group was not linked to the carboxyl group directly. It was thus tentatively identified as polygalacic acid 3-O-β-d-glucopyranoside and peak 45 was tentatively identified as 19α-hydroxyasiatic acid-28-O-β-d-glucopyranoside [34]. Peaks 46, 50 and 52 with m/z 649.39552 (C36H58O10), 503.33973 (C30H48O6) and 487.34276 (C30H48O5) were tentatively identified as kajiichigoside F1, 1-hydroxyeuscaphic acid, and euscaphic acid, respectively, according to previous reports [7,10,14]. Peaks 51 and 53 had the same molecular formula of C30H46O5. Peak 53 lost neutral ions of H2O and CO2, then produced fragment ions of m/z 467 and 441 in MS spectrum, indicating the presence of hydroxyl and carboxyl groups. Therefore, peak 53 was identified as 2α,19α-dihydroxy-3-oxours-12-en-28-oic acid [34,35], and peak 51 was its isomer. Peaks 54 and 55 gave the same molecular formula and fragment ions in their MS spectra, and they were tentatively identified as pomolic acid or an isomer, which needs to be further confirmed by nuclear magnetic resonance (NMR) [7,10,14]. Peak 56 gave a [M − H]− ion at m/z 469.33187 (C30H46O4). The fragment ions at m/z 451 and 407 were produced by the loss of H2O and the subsequent loss of CO2, and it was therefore tentatively identified as 2α,3β-dihydroxylup-20(29)-en-28-oic acid [34].

Amino acids: Nine amino acids identified in the two fruits have been confirmed with reference standards, and they were serine (2), arginine (3), proline (4), valine (7), tyrosine (11), isoleucine (13), leucine (14), phenylalanine (17) and tryptophan (20) [18].

Phenylpropanoid derivatives: Peaks 26 and 41, a pair of isomers, gave a [M + H]+ ion at m/z 407.16 (C21H26O8) and the fragment ion of m/z 245, 162 Da lost from the precursor ion, corresponding to a glucose group. Thus, they were identified as erythro-guaiacylglycerol β-sinapyl ether and threo-guaiacylglycerol β-sinapyl ether, but the configuration of the chiral carbon cannot be confirmed without NMR [26]. Peak 33 gave a [M − H]− ion at m/z 417.15509, with a molecular formula C22H26O8 by TOF–MS. The fragment ion at m/z 181 was produced by the dissociation of the furan ring; it therefore was identified as diasyringaresinol [33]. Peak 39 showed a [M − H]− ion at m/z 435.12995 (C21H24O10). The fragment ion of m/z 273 unequivocally illustrated the existence of a glucose group. Thus it was tentatively identified as phlorizin [36]. Peak 42 with a protonated [M + H]+ ion at m/z 359.14831 (C20H22O6) was identified as pinoresinol compared with a previous report [36].

Condensed tannins: In the MS spectra, four condensed tannins were identified on the basis of a fragmentation pattern with successive loss of 288 Da corresponding to the loss of catechin units (C15H12O6) [37]. According to previous reports [35,36], they were tentatively identified as procyanidin B1 (21), procyanidin B2 (24), fisetinidol-(4α,8)-catechin (27) and procyanidin B3 (29) with the depronated ions at m/z 305.06686, m/z 577.13504, m/z 577.13567, m/z 561.14044 and m/z 577.13543, respectively.

Miscellaneous compounds: Peak 15 was exactly identified as vanillin by comparison with a reference standard [33]. Peak 22 exhibited a deprotonated ion at m/z 181.05076 corresponding to molecular formula of C9H10O4. The fragment ions at m/z 163, 135 and 119 indicated the existence of hydroxyl, carbonyl and methoxyl groups. It was therefore identified as syringaldehyde [33]. Peak 37 displayed a [M − H]− ion at m/z 389.12421 (C20H22O8), and the product ion of m/z 273 hinted the presence of glucose group. It was tentatively identified as piceid [36]. Peak 40 with C15H12O2 gave fragments at m/z 197 and 185 in its MS spectrum, which were generated by losing CH2O and subsequently H2O, and was identified as 3-methoxy-5-hydroxy-stilbene [36]. Peak 23 was 162 Da more than peak 25, and the product ions were very similar to peak 25. Thus it was identified as 4-hydroxybenzoic acid-4-O-β-d-glucopyranoside [36].

2.2.2. Comparison of Multiple Constituents

UFLC/Q-TOF-MS has become a popular method to analyze constituents in complex systems due to the provided precise molecular weight and fragment characteristic information. Chemical profiles of R. roxburghii and R. sterilis fruits showed obviously differences based on the representative negative signals (Figure 3). The structures of identified compounds were shown in Figure 4.

Figure 3

The representative total ion chromatograms of R. roxburghii (A) and R. sterilis (B) fruits obtained from UFLC/Q-TOF-MS in negative ion mode.

Figure 4

Chemical structures of compounds identified in the methanol extracts of R. roxburghii and R. sterilis fruits.

Organic acids were the main chemotypes reported in R. roxburghii fruit, especially its high ascorbic acid level. Huang et al. demonstrated that the content of ascorbic acid in R. roxburghii fruit is higher than that in most common fruit crops, e.g., tomato (~20 mg), strawberry (~50 mg), and kiwifruit (~100 mg) [38]. In this study, a high content of ascorbic acid was also found and determined by a LC-MS method [39]. Comparing the difference of organic acid compositions between R. roxburghii and R. sterilis fruits, compound 57, namely 9,12,15-octadecatrienoic acid was only detectable in R. sterilis fruit, which was consistent with the result of GC-MS; whereas, syringic acid (19) was only found in R. roxburghii fruit rather than in R. sterilis fruit.

Flavonoids were the second main constituents in both R. roxburghii and R. sterilis fruits. The radioprotective effects of flavonoids from R. roxburghii fruit has been proved by cell model and animal experiments [5]. There were 11 flavonoids, including quercetin, kaempferol and their derivatives found in R. roxburghii and R. sterilis fruits. However, epigallocatechin (18) was only found in R. sterilis fruit rather than in R. roxburghii fruit.

More than twenty triterpenes have so far been previously found in the Rose family [34]. Triterpenes were other main constituents in R. roxburghii fruit with α-glucosidase inhibitory activity [40]. Liang et al. [14] reported that four kinds of triterpenes were isolated from R. sterilis fruit three decades ago. In this study, eleven triterpenes were shared between R. roxburghii and R. sterilis fruits. In addition, polygalacic acid 3-O-β-d-glucopyranoside (43), 19a-hydroxyasiatic acid-28-O-β-d-glucopyranoside (45), 2α,19α-dihydroxy-3-oxo-urs-12-en-28-oic acid (53), 2α,3β-dihydroxylup-20(29)-en-28-oic acid (56) and ursolic acid (58) were reported for the first time in R. sterilis fruit.

Amino acids as nutrients have various functions in human beings. In addition, the amino acid composition plays an important role affecting the flavor of fruits. In term of the types of compounds, there was no difference between R. roxburghii and R. sterilis fruits [18].

A total of five phenylpropanoid derivatives were identified in R. roxburghii and R. sterilis fruits. Among them, diasyringaresinol (33) and phlorizin (39) were only found in R. roxburghii fruit.

Tannins are a kind of secondary metabolite common reported in the genus Rosa, and most of them are characterized by catechin and epicatechin as constitutive units. For example, Yan et al. [36] reported five condensed tannins in R. laevigata, namely procyanidin B3, fisetinidol-(4α,8)-catechin, guibourtinidol-(4α,8)-catechin, ent-isetinidol-(4α,6)-catechin, fisetinidol-(4β,8)-catechin. In this study, procyanidin B1 (21), procyanidin B2 (24), and procyanidin B3 (29) were commonly found in R. roxburghii and R. sterilis fruits. Interestingly, fisetinidol-(4α,8)-catechin (27) was only found in R. sterilis fruit.

Five miscellaneous compounds, including two benzaldehyde derivatives (15 and 22), two stilbenes (37 and 40) and one benzoic acid derivative (23), were detected in this study. Except for the two stilbenes, the presence of the three compounds in R. roxburghii and R. sterilis fruits was different although they have been found in Rosa species before. Vanillin (15) and syringaldehyde (22) were only detected in R. roxburghii fruit and 4-hydroxybenzoic acid-4-O-β-d-glucopyranoside (23) only existed in R. sterilis fruit.

Generally, 50 phytochemicals existed in both R. roxburghii and R. sterilis fruits. Five characteristic compounds, including vanillin (15), syringic acid (19), syringaldehyde (22), diasyringaresinol (33) and phlorizin (39) were found in R. roxburghii fruit and the other four compounds, including epigallocatechin (18), 4-hydroxybenzoic acid-4-O-glucopyranoside (23), fisetinidol-(4α,8)-catechin (27), and 9,12,15-octadecatrienoic acid (57) were only detectable in R. sterilis fruit. The nine characteristic phytochemicals were considered to be potential markers for discriminating R. roxburghii fruit from R. sterilis fruit in quality control. It is general known that phytochemicals are responsible for the bioactivities of medicinal plants. Clarifying the characteristic phytochemicals in R. roxburghii and R. sterilis fruits is very important for their development and application in health-related industries.

3. Materials and Methods

3.1. Chemicals and Reagents

Methanol (HPLC grade) and n-hexane (GC grade) were purchased from Merck KGaA (Darmstadt, Germany). Distilled water was from Guangzhou Watson’s Food & Drinks Co., Ltd. (Guangzhou, China). The other reagents used were of analytical grade and were used without any further purification. Gallic acid, 4-hydroxybenzoic acid, caffeic acid, protocatechuic acid, isoquercetin, quercitrin, quercetin and kaempferol were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Lactic acid, malic acid, ascorbic acid, citric acid, tryptophan, rutin and luteolin were purchased from Aladdin Industrial Corporation (Shanghai, China). Serine, arginine, proline, valine, tyrosine, leucine, isoleucine, phenylalanine, p-coumaric acid, vanillin and syringic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). n-Hexadecanoic acid, 9,12,15-octadecatrienoic acid, 9,12-octadecadienoic acid, and 9-octadecenoic acid were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). The purities of all compounds were over 95%, as determined by the area normalization method using HPLC or GC.

3.2. Plant Material

R. roxburghii and R. sterilis were cultivated in Guizhou province and their fruits for each species were collected from five cultivars in July (R. roxburghii) and October (R. sterilis), 2015, respectively. All of the specimens were exactly identified by the authors and stored in −80 °C in Guangzhou Institute of Advanced Technology, Chinese Academy of Sciences, China.

3.3. GC-MS Experiment

3.3.1. Sample Preparation

The fresh fruit was washed three times with distilled water and cut randomly into small pieces. Then 100 g of the chipped sample (20 g from each cultivar) was stirred in a bullet blender (Jiuyang Co., Ltd., Shandong, China) for 1 min. After adding 200 mL distilled water, the sample was subjected to hydrodistillation in a steam distillation vessel for 3 h. The oil was extracted by 2 mL hexane and dried with excess anhydrous sodium sulphate. Finally, the oil extract was filtered by 0.22 μm microporous membrane and stored at 4 °C before injection [41].

3.3.2. Instrument Conditions

The analysis was carried out with an 7890A-5975C GC-MSD instrument (Agilent, Santa Clara, CA, USA). Separations were performed using an Agilent HP-5MS capillary column (30 m × 250 mm × 0.25 μm). The GC oven temperature was programmed from 50 °C (1 min isothermal) to 160 °C at 15 °C·min−1, and then to 280 °C at 5 °C·min−1 (5 min isothermal). A 5 μL sample solution was injected into GC system without split. Helium was the carrier gas with 2 mL/min. The injector temperature was set to 280 °C. The MS was operated in full scan mode (50–500 amu at 0.5 scan/s) with electron ionization mode at 70 eV.

3.3.3. Data Analysis

Data were evaluated by MSD ChemStation E.02.02.1431 software (Agilent). The identification of the compounds was carried out by comparing with data known from the literature, the NIST 05 library (NIST Mass Spectral Database, PC-Version 5.0, 2005, National Institute of Standards and Technology, Gaithersburg, MD, USA) and authentic standards. Percentage data of the total ion current chromatograms were calculated by the area normalization method without applying response factor correction and shown as mean ± standard deviation (SD).

3.4. UFLC/Q-TOF-MS Experiments

3.4.1. Sample Preparation

Three hundred gram of fresh fruits (60 g from each cultivar) were washed three times with distilled water and cut into small pieces (thickness about 0.1 cm) and then lyophilized by Modulyo vacuum freeze-drying (Thermo Fisher Scientific Inc., Waltham, MA, USA). During the whole freeze-drying process, the temperature was kept at −50 °C with vacuum degree of 10 mbar for 48 h. Then the dried samples were crushed by Media BM252C blender (Midea Group Ltd., Guangdong, China) and stored at −20 °C until use. One gram of the dried fine powder was accurately weighed and extracted with 25 mL methanol twice in a KQ600DE ultrasonic bath (Kunshan, Jiangsu, China) for 30 min at room temperature. After filtered, the combined extract was dilute with methanol to 50 mL and stored at 4 °C for further study.

3.4.2. Instrument Conditions

The multiple constituents in the methanol extraction of R. roxburghii and R. sterilis fruits were identified by UFLC/Q-TOF-MS method. Chromatographic analysis was performed on a UFLC XR system (Shimadzu Corporation, Kyoto, Japan) equipped with a LC-20AD-XR binary pump, SIL-20AD-XR autosampler and a CTO-20A column oven. The column was an Agilent Eclipse Plus C18 (2.1 mm × 100 mm, 1.8 μm, Agilent), maintained at 35 °C. The sample was eluted at a flow rate of 0.2 mL/min in a linear gradient mode of A (0.1% formic acid:water) and B (0.1% formic acid:methanol): 0–40 min (5%–100% B), then kept for 3 min at 100% B. The injection volume was 5 μL. Mass spectrometry was performed on the triple TOF™ 5600 (AB SCIEX, Foster City, CA, USA) a hybrid triple quadrupole time-of-flight mass spectrometer equipped with ESI source, and mass range was set at m/z 100–1200. The experiment parameters were as follows: CUR: 35; GS1: 55; GS2l 55; ISVF: 4500; TEM: 550. Nitrogen was used as nebulizer and auxiliary gas. All the acquisition and analysis of data were controlled by the Peak View Software TM V.1.6 (AB SCIEX).

3.4.3. Data Analysis

Compound identification of methanol extracts of R. roxburghii and R. sterilis fruits was carried out by comparison of MS and collision-induced dissociation (CID) spectral data of analytes with those of reference standards. When standard substances were unavailable, compounds were tentatively identified by precise molecular weight within an accuracy of 5 ppm or less which can be uniquely associated with a specific molecular formula. At the same time, based on the characteristic pyrolysis fragments obtained by CID spectra, compound structures were assigned by comparison with literature data and standards with similar structures.

4. Conclusions

This study analyzed and compared the important dietary constituents in R. roxburghii and R. sterilis fruits, including essential oils, organic acids, flavonoids, triterpenes, amino acids, phenylpropanoid derivatives, condensed tannins, stilbenes, benzaldehyde derivatives and a benzoic acid derivative. The phytochemical profiles and the chemical differences of these two fruits have been generally illustrated. However, a series of R. roxburghii and R. sterilis fruits, as the various samples, should be collected from the different producing areas for further study. The contents of main constituents and characteristic compounds, along with their bioactivities, need further in-depth study.