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. 2016 Sep 9;21(9):1204. doi: 10.3390/molecules21091204

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

Meng-Hua Liu 1, Qi Zhang 1, Yuan-He Zhang 2, Xian-Yuan Lu 1, Wei-Ming Fu 1,2,*, Jing-Yu He 2,*
Editor: Derek J McPhee
PMCID: PMC6272847  PMID: 27618004

Abstract

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.

Keywords: Rosa roxburghii, Rosa sterilis, constituents, GC-MS, UFLC/Q-TOF-MS

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.

Figure 1

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

Figure 2.

Figure 2

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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
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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.

Figure 3

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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.

Figure 4

Figure 4

Figure 4

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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.

Acknowledgments

The study was supported by the Open Project of Guangdong Provincial Key Laboratory of New Drug Screening, the PhD Start-up Fund of Natural Science Foundation of Guangdong Province (2015A030310139) and the Science and Technology Program of Guangzhou (2014J4100045).

Author Contributions

Jing-yu He and Wei-ming Fu conceived and designed the experiments; Meng-hua Liu, Qi Zhang, Yuan-he Zhang and Jing-yu He performed the experiments; Meng-hua Liu and Qi Zhang analyzed the data; Meng-hua Liu and Xian-yuan Lu wrote the manuscript; Jing-yu He and Wei-ming Fu modified the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Sample Availability: Samples of the compounds are available from the authors.

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