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.


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 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.
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. 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. 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 antiinflammatory 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.
Phenylpropanoid derivatives: Peaks 26 and 41, a pair of isomers, gave a [M + H] + ion at m/z 407.16 (C 21 H 26 O 8 ) 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 C 22 H 26 O 8 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 (C 21 H 24 O 10 ). 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 (C 20 H 22 O 6 ) 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 (C 15 H 12 O 6 ) [37]. According to previous reports [35,36], they were tentatively identified as procyanidin B1 (21), procyanidin B2 (24) 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 C 9 H 10 O 4 . 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 (C 20 H 22 O 8 ), and the product ion of m/z 273 hinted the presence of glucose group. It was tentatively identified as piceid [36]. Peak 40 with C 15 H 12 O 2 gave fragments at m/z 197 and 185 in its MS spectrum, which were generated by losing CH 2 O and subsequently H 2 O, 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].

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. 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) 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].

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.     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.
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.
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 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.
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.

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.