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Flavonol glycosides in the petal of Rosa species as chemotaxonomic markers
Phytochemistry 107 (2014) 61–68
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Flavonol glycosides in the petal of Rosa species as chemotaxonomic markers Ochir Sarangowa a,1, Tsutomu Kanazawa a, Makoto Nishizawa b, Takao Myoda b, Changxi Bai c, Takashi Yamagishi a,⇑ a b c
Kitami Institute of Technology, 165 Koen-cho, Kitami 090-8507, Japan Faculty of Bio-Industry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan Academy of Mongolian Medicine, Inner Mongolia Medical University, Hohhot 010110, China
a r t i c l e
i n f o
Article history: Received 22 March 2014 Received in revised form 30 July 2014 Available online 11 September 2014 Keywords: Rosa species Rosa gallica Rosa rugosa Rosaceae Quercetin glycoside Kaempferol glycoside Chemotaxonomy
a b s t r a c t Thirteen flavonol glycosides were isolated from the petals of Rosa species belonging to the section Gallicanae, and their structures were identified from their spectroscopic data. These flavonol glycosides, along with two flavonol glycosides isolated from Rosa rugosa, in the petals of 31 Rosa species belonging to sections Gallicanae, Cinnamomeae, Caninae, and Synstylae were quantitatively analyzed by UPLC. The results indicated that the species belonging to these sections could be classified into four types (Type A, B, C and D) based on the pattern of flavonol glycoside contents, whereas the R. rugosa flavonol glycosides were detected only in section Cinnamomeae. A principal components analysis (PCA) calculated from the 15 flavonol glycosides contained in these samples supported the presence of four types. The distribution of the species in Type D (a group of Cinnamomeae) was shown to reflect close interrelationships, but species in Type B (one group of Gallicanae) could be subdivided into two groups, one of which contained species in section Synstylae. Moreover, the flavonol glycosides were grouped by sugar moieties: a disaccharide composed of two hexoses (S1), a hexose (S2), including a hexose with galloyl group, a pentose (S3), and a disaccharide composed of a hexose and a pentose (S4). The ratios of the amounts of S1–S4 to total flavonol glycoside content indicated that differences among the four sections were more distinctive than the amounts of the 15 flavonol glycosides. The 31 samples were divided into Type B, composed of one type of Gallicanae and Synstylae, Type A + C, composed of another type of Gallicanae and Caninae, and Type D, composed of Cinnamomeae. The R. rugosa flavonol glycosides were shown to be important chemotaxonomic markers for the classification of species in Cinnamomeae, and this method of using flavonol glycosides as chemotaxonomic markers could be useful for the identification of Rosa species belonging to sections Gallicanae, Cinnamomeae, Caninae, and Synstylae. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Petals and blossoms of roses have been used for medicines and as an ingredient of herbal tea in eastern Asia. As the original rose plants differ depending on country, the aim herein was to characterize original plants by phytochemical methods. In Japan, Rosa rugosa, a wild rose distributed mainly in the northern islands, has been used for an herbal tea (Gault and Synge, 1971). In China, roses used for medicines and in herbal teas ⇑ Corresponding author. Address: Applied Research Center, Kitami Institute of Technology, 165 Koen-cho, Kitami 090-8507, Japan. Tel.: +81 157 26 9462; fax: +81 157 26 9461. E-mail address: [email protected] (T. Yamagishi). 1 Present address: Academy of Mongolian Medicine, Inner Mongolia Medical University, Hohhot 010110, China. http://dx.doi.org/10.1016/j.phytochem.2014.08.013 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
are called ‘‘Mei-gui Hua’’, but many kinds of Mei-gui Hua prepared from different plant species are commercially available (Gu and Kenneth, 2003). In the Xinjiang province, the westernmost part of China, Mei-gui Hua made from the blossoms or petals of roses cultivated there are called ‘‘Kizil gul’’, and they have been used in herbal teas for the prevention and treatment of diabetes (Muhammet, 2002). In a previous paper, morphological, phylogenic and phytochemical studies established that the plant species of Mei-gui Hua cultivated in the Xinjiang province must be Rosa gallica or its hybrids, whereas those cultivated in the northeastern part of China are hybrids of R. rugosa. In phytochemical studies, the 3-O-b-D-glucosides of quercetin and kaempferol were not detected in petals of wild R. rugosa collected in Japan and Korea. However, the 3-O(200 -O-b-D-glucosyl)-b-D-xylosides of quercetin and kaempferol
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were detected in the petals of R. rugosa, but were not detected in Mei-gui Hua cultivated in the Xinjiang province. There have also been some reports on the characterization of sections of subgenus Rosa using flavonol glycosides, thus the content of flavonol glycosides may be useful to characterize plant species belonging to sections of subgenus Rosa for medicinal use (Sarangowa et al., 2010). Therefore, the content of 15 flavonol glycosides (Fig. 1) are quantitatively determined in the petals of roses belong to sections Gallicanae and Cinnamomeae. In this paper, the usefulness of flavonol glycosides for chemotaxonomy of these sections belonging to subgenus Rosa are reported.
hexose, and the spectra of 12 and 13 showed the presence of a pentose. Comparing the 1H and 13C NMR data and MS data of these compounds with literature values (Nowak and Wolbis, 2002; Alaniya et al., 2012; Park et al., 2012), the structures of 8, 12 and 13 were characterized as kaempferol 3-O-b-D-galactoside (8), kaempferol 3-O-a-L-arabinofuranoside (12), and kaempferol 3-Oa-D-rhamnoside (13). Quercetin 3-O-(200 -O-galloyl)-b-D-glucoside (6) and kaempferol 3-O-(200 -O-galloyl)-b-D-glucoside (7) were also isolated from R. gallica as detailed in our previous report (Sarangowa et al., 2013) (Fig. 1).
2. Results
2.2. Quantitative determination of flavonol glycosides in sections Gallicanae, Cinnamomeae, Caninae and Synstylae
2.1. Isolation of flavonol glycosides from R. gallica The 50% EtOH extracts of the petals of a hybrid of R. gallica collected in the Xinjiang province in China were continuously partitioned with a H2O–MeOH–CHCl3 system, and the upper layer was fractionated by column chromatography on MCI gel CHP-20P followed by chromatography on Sephadex LH-20. The fractions containing flavonol glycosides were further purified using a reversed phase gel (Wakosil 40 C18) to give compounds 1–13. Compounds 1, 2, 3, 5 and 9 were isolated as the major flavonol glycosides in the petals of R. gallica, and were identified as quercetin 3-O-(200 O-b-D-glucosyl)-b-D-galactoside (1), quercetin 3-O-sophoroside (2), kaempferol 3-O-sophoroside (3), quercetin 3-O-b-D-glucoside (5) and kaempferol 3-O-b-D-glucoside (9), respectively, as previously described (Sarangowa et al., 2010) and by comparison of their 1H and 13C NMR data and MS data with literature values (Iwashina et al., 2005; Schliemann et al., 2006; Ross et al., 2005). The 1H NMR spectra of 4, 10 and 11 indicated the presence of quercetin as an aglycone. The 13C NMR spectrum of compound 4 showed the presence of a hexose, and the spectra of 10 and 11 showed the presence of a pentose. Comparing the 1H and 13C NMR data and MS data with literature data (Lin and Lin, 1999; Nowak and Wolbis, 2002; Park et al., 2012; Tanaka et al., 1981; Zhu et al., 2013), the structures of 4, 10 and 11 were identified as quercetin 3-O-b-D-galactoside (4), quercetin 3-O-b-D-xylopyranoside (10) and quercetin 3-O-a-L-arabinofuranoside (11), respectively. The positions of the sugar moiety on quercetin were confirmed by analysis of HMBC spectra. The C1–H of the sugar moiety gave a cross peak with the C3 position of quercetin. The 1H NMR spectra of compounds 8, 12 and 13 indicated presence of kaempferol as the aglycone (Xiao et al., 2006; Zhu et al., 2013). The 13C NMR spectrum of 8 showed the presence of a
glycosides of quercetin 1: R= 3-O-(2"-O-β-D-glucosyl)-β-D-galctoside 2: R= 3-O-sophoroside 4: R= 3-O-β-D-galctoside 5: R= 3-O-β-D-glucoside 6: R=3-O-(2"-O-galloyl)-β-D-glucoside 10: R= 3-O-β-D-xylopyranoside 11: R= 3-O-α-L-arabinofuranoside 14: R= 3-O-(2"-O-β-D-glucosyl)-β -D-xyloside
A method using ultra-performance liquid chromatography (UPLC) equipped with a photodiode array (PDA) detector was optimized for quantitative determination of flavonol glycosides in the 50% EtOH extract of the petal of 31 Rosa species (Table 1). The monitoring wavelength was set at 350 nm, which was the absorption maximum for glycosides of quercetin and kaempferol. Chromatography peaks of quercetin and kaempferol glycosides were identified from their retention times and UV spectra monitored by PDA detector. The content of flavonol glycosides was quantitatively analyzed by UPLC. Typical chromatograms of hybrids of R. gallica and R. rugosa are shown in Fig. 2. Seven glycosides of quercetin (1, 2, 4, 5, 6, 10 and 11) and six glycosides of kaempferol (3, 7, 8, 9, 12 and 13) were detected in the chromatogram of the hybrid of R. gallica. However, in the chromatogram of R. rugosa, 3-O-(200 -O-b-D-glycosyl)b-D-xylosides of quercetin (14) and kaempferol (15) were detected along with 2 and 3, while other glycosides were not detected. The content of fifteen glycosides of quercetin and kaempferol are shown in Table 2. Fifteen flavonol glycosides were identified in this experiment; however, all of the flavonol glycosides were not detected in all samples. The total content of these glycosides was highest in the petals of Rosa species in section Gallicanae (28.68–119.10 mg/g), followed by section Caninae (30.78 and 41.52 mg/g) and section Synstylae (30.49 and 48.90 mg/g). The glycoside content in the petals of Rosa species in Section Cinnamomeae was lower than the other sections (0.48–7.5 mg/g). Flavonol glycosides including a hexose (4, 5, 8, 9 and 13) and their galloyl derivatives (6 and 7), and those with a pentose (10, 11 and 12) were detected only in section Gallicanae, except for Rosa acicularis (Ci05), Rosa davurica (Ci08) and R. rugosa Rosa rubrifoia
glycosides of kaempferol 3: R= 3-O-sophoroside 7: R= 3-O-(2"-O-galloyl)-β-D-glucoside 8: R= 3-O-β-D-galctoside 9: R= 3-O-β-D-glucoside 12: R= 3-O-α-L-arabinofuranoside 13: R= 3-O-α-D-rhamnoside 15: R= 3-O-(2"-O-β-D-glucosyl)-β-D-xyloside
Fig. 1. Glycosides of quercetin and kaempferol.
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O. Sarangowa et al. / Phytochemistry 107 (2014) 61–68 Table 1 List of samples. Sample code
Species
Collection reference
Date collected
Section Gallicanae Ga01-1 Ga01-2 Ga01-3 Ga02 Ga03 Ga04 Ga05 Ga06 Ga07 Ga08 Ga09 Ga10 Ga11 Ga12
R. R. R. R. R. R. R. R. R. R. R. R. R. R.
gallica (hybrid) gallica (hybrid) gallica (hybrid) gallica var. officinalis Ser. gallica ‘Complicate’ gallica ‘Alika’ gallica ‘Charles de Mills’ gallica ‘OeilletFlamand’ gallica ‘James Mason’ gallica ‘Francofurtana’ centifolia var. sancta (Richard) Zabel damascena ‘Bifera’ damascena ‘Perpetual’ damascena ‘Jacques Cartier’
Farm-raised Farm-raised Farm-raised Commercial Commercial Commercial Commercial Commercial Commercial Preservation Commercial Commercial Commercial Commercial
02/05/2007 01/05/2007 01/05/2007 06/07/2010 25/06/2010 25/06/2010 25/06/2010 25/06/2010 25/06/2010 28/09/2010 06/07/2011 02/07/2012 02/07/2012 13/08/2009
Section Cinnamomeae Ci01-1 Ci01-2 Ci01-3 Ci01-4 Ci01-5 Ci01-6 Ci02 Ci03 Ci04 Ci05 Ci06-1 Ci06-2 Ci06-3
R. R. R. R. R. R. R. R. R. R. R. R. R.
rugosa Thunb. rugosa Thunb. rugosa Thunb. rugosa Thunb. rugosa Thunb. rugosa Thunb. rugosa R. rubrifolia rugosa var. rubra Rehder maikwai Hara acicularis Lindl. davurica Pall.var. alpestris (Nakai) Kitag. davurica Pall.var. alpestris (Nakai) Kitag. davurica Pall.var. alpestris (Nakai) Kitag.
Wild Wild Wild Wild Wild Wild Preservation Commercial Preservation Preservation Preservation Wild Commercial
26/08/2007 05/05/2009 06/07/2010 06/07/2010 06/07/2010 06/07/2010 02/07/2011 25/06/2010 06/07/2010 06/07/2010 18/06/2011 29/06/2007 25/06/2010
Section Caninae Ca01 Ca02
R. rubrifolia Vill. (R. glauca Pourr.) R. canina L.
Preservation Commercial
02/07/2011 25/06/2010
Section Synstylae Sy01 Sy02
R. multiflora Thunb. R. gentileana Léveillé & Vaniot
Preservation Commercial
02/07/2011 25/06/2010
Ga01-1: Hotan, Xiyumeigui farm, Xinjiang province, China. Ga01-2: Niya, Xinjiang Province, China. Ga01-3: Keriye, Xinjiang Province, China. Ga02, Ga09 and Ga12: Chippubetsu Rose Garden, Chippubetsu, Hokkaido, Japan. Ga03, Ga04, Ga05, Ga06, Ga07, Ga10, Ga11, Ci03, Ci06-3 Ca02 and Sy02: Yurigahara Lily Park, Sapporo, Japan. Ga08: The Research Center for Medicinal Plant Resources National Institute of Biomedical Innovation, Nayoro, Hokkaido, Japan. Ci01-1: Komuke Natural Flower Garden, Monbetsu, Hokkaido, Japan. Ci01-2: Sado Island, Niigata, Japan. Ci01-3: Yangyang, Gangwon-do, Korea. Ci01-4: HwasongSongsan, Gangwon-do, Korea. Ci01-5: Pingying, Shandong Province, China. Ci02, Ci05, Ci06-1, Ca01 and Sy01: Hokkaido Forestry Research Institute, Bibai, Hokkaido, Japan. Ci04: Experimental Station for Medicinal Plant Studies, Hokkaido University, Sapporo, Japan. Ci06-2: Kamikawa, Hokkaido, Japan. All samples are kept in The Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation, Nayoro, Hokkaido, Japan (Yamagishi crude drag library, code No. Ga01-1, Ga01-2, Ga01-3, Ga02, Ga03, Ga04, Ga05, Ga06, Ga07, Ga08, Ga09, Ga10, Ga11, Ga12, Ci01-1, Ci01-2, Ci01-3, Ci01-4, Ci01-5, Ci01-6, Ci06-1, Ci06-2, Ci06-3, Ca01, Ca02, Sy01, Sy02 Ci02, Ci03, Ci04, Ci05).
(Ci02). On the other hand, the Rosa species in section Cinnamomeae mainly contained glycosides with a disaccharide (1, 2, 3, 14 and 15). Sophorosides of quercetin and/or kaempferol (2 and 3, respectively) were detected in almost all of the samples, and two patterns involving the presence and content of these glycosides along with the content of 1 were recognized. Samples belong to pattern 1 contained relatively large amount of 1, 2 and 3, and those belonging to pattern 2 contained lower amounts of these compounds (or were not detected). Compounds 14 and 15 were detected only in section Cinnamomeae. 3. Discussion
collected in the Xinjiang province, China. In the present study, samples of Rosa plants were collected at the same location for analysis. As reported previously, the roses used as ‘‘Mei-gui’’ or ‘‘Kizil gul’’ in Xinjiang province are not R. rugosa, but are R. gallica and its hybrids. In this paper, glucosides 4, 9, 11 and 12 were not detected in 6 samples of R. rugosa collected in Japan, Korea and China, and 1, 3, 4, 9, 11 and 12 were detected in all of the samples collected in Xinjiang province. Therefore, the plants collected by Xiao et al. (2006) were supposed to be hybrids of R. gallica. In this study, additional glycosides of quercetin and kaempferol including 2, 5, 8, 10 and 13 were isolated for the first time from hybrids of R. gallica.
3.1. Flavonol glycosides in R. gallica
3.2. Flavonol glycosides as chemotaxonomic markers
Xiao et al. (2006) reported the isolation of glycosides of kaempferol and quercetin such as 1, 3, 4, 9, 11 and 12 from ‘‘R. rugosa’’
Mikanagi et al. (1990, 1995) reported that quercetin 3O-glucoside (5) and kaempferol 3-O-glucoside (9) were the major
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Fig. 2. UPLC Chromatograms of the 50% EtOH extract of the petal. a: R. gallica b: R. rugosa. 1, quercetin 3-O-(200 -O-b-D-glucosyl)-b-D-galactoside; 2, quercetin 3-O-sophoroside; 3, kaempferol 3-O-sophoroside; 4, quercetin 3-O-b-D-galactoside; 5, quercetin 3-O-b-D-glucoside; 6, quercetin 3-O-(200 -O-galloyl)-b-D-glucoside; 7, kaempferol 3-O-(200 -Ogalloyl)-b-D-glucoside; 8, kaempferol 3-O-b-D-galactoside; 9, kaempferol 3-O-b-D-glucoside; 10, quercetin 3-O-b-D-xylopyranoside; 11, quercetin 3-O-a-L-arabinofuranoside; 12, kaempferol 3-O-a-L-arabinofuranoside; 13, kaempferol 3-O-a-D-rhamnoside; 14, quercetin3-O-(200 -O-b-D-glucosyl)-b-D-xyloside; 15, kaempferol 3-O-(200 -O-b-D-glucosyl)b-D-xyloside.
flavonoid glycosides in section Gallicanae. In this study, flavonol glycosides in the hybrids of R. gallica were analyzed, and compounds 5 and 9 were detected in all samples. However, the results indicated that samples in this section could be divided into three types according to the content level of these flavonol glycosides. Type A was composed of three samples (Ga01-1–Ga01-3) collected in the Xinjiang province in China, containing 1, 2, 3, 4 and 13 in high levels along with 5 and 9. Type B was composed of nine samples (Ga02–03, Ga05–06 and Ga08–12), containing 5 and 9 as the major flavonol glycosides, but the content of 2 and 3 was low, and 1 was not detected. Type C was composed of two samples (Ga04 and Ga07), containing 1, 2 and 3 in high content, and 5 and 9 were in low content. Contrary to Mikanagi’s reports, these results showed that compounds 5 and 9 were not necessarily the major flavonol glycosides, which indicated that these samples were hybrids of R. gallica. In fourteen samples of Gallicanae, 9 was detected in all of the samples, but the contents of 5 was low in some samples. These results here indicated that a notable feature of Rosa species in section Cinnamomeae was their low contents of flavonol glycosides (0.48–7.50 mg/g). The average total contents of flavonol glycosides (3.19 ± 2.26 mg/g) was more than twenty fold lower for Cinnamomeae than for Gallicanae (67.09 ± 23.31 mg/g). Mikanagi et al. (1995) and Grossi et al. (1998) reported that quercetin 3-O-sophoroside (2) and kaempferol 3-O-sophoroside (3) were characteristic markers of the section Cinnamomeae. In the results here, compound 2 was detected in all Cinnamomeae
samples, but compound 3 was not detected in all samples, and some samples contained compound 1. On the other hand, these results indicated that the more characteristic flavonol glycosides were the 3-O-(20 -O-b-D-glycosyl)-b-D-xyloside of quercetin (14) and kaempferol (15). This characteristic feature of section Cinnamomeae was defined as Type D, as there were no samples in Gallicanae, Caninae, or Synstylae that contained 14 and 15. Therefore, 14 and 15 could be important chemotaxonomic markers of section Cinnamomeae. Grossi et al. (1998) have also reported that section Synstylae was well defined by the presence of kaempferol 3-O-glucoside (9) and kaempferol 3-O-rhamnoside (13). The results here were consistent with their report, as compound 9 was the major flavonol glycoside followed by 13 identified in this subspecies. There was no characteristic feature of flavonol glycoside content in the samples in section Caninae and Synstylae, which was similar to those of Type B and Type C of section Gallicanae, respectively. The total contents of flavonol glycosides in these three sections were higher than those in section Cinnamomeae. Principal component analysis (PCA) of the content of fifteen glycosides of quercetin and kaempferol in 31 Rosa species was performed using SPSS Statistics software. As shown in Fig. 3, the distribution plots were divided into five groups. In the third quadrant, thirteen species in section Cinnamomeae (Type D) were closely distributed. This close distribution of the samples in Cinnamomeae must be due to the samples in section Cinnamomeae all being wild or preservation plants. In contrast to
Table 2 Contents of flavonol glycosides in the petals of Rosa species. Section
Code No.
Content of flavonol glycosidesa (mg/g D.W.)
Total
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Ga01-1 Ga01-2 Ga01-3 Ga02 Ga03 Ga04 Ga05 Ga06 Ga07 Ga08 Ga09 Ga10 Ga11 Ga12
13.57 11.02 13.56 0.00 0.00 13.72 0.00 0.00 11.82 0.00 0.00 0.00 0.00 0.00
13.14 11.81 14.08 0.03 0.03 12.17 0.23 0.29 8.79 0.07 0.12 0.06 0.08 0.00
8.07 7.95 8.91 0.25 0.10 20.95 0.22 0.12 11.56 0.25 1.23 0.01 0.00 0.00
6.57 6.18 7.21 12.41 4.52 1.38 17.89 23.20 1.12 16.15 4.05 3.97 3.86 6.44
11.37 11.31 12.25 10.96 9.95 1.79 22.42 10.49 1.23 19.15 8.06 3.89 5.69 7.91
0.05 0.04 0.05 0.11 0.10 0.02 0.04 0.12 0.02 0.04 0.01 0.09 0.06 0.12
0.06 0.07 0.09 0.05 0.01 0.02 0.11 0.12 0.00 0.10 0.02 0.03 0.02 0.07
2.87 3.18 3.72 11.76 1.03 2.35 6.36 13.63 2.00 4.30 3.12 5.05 2.82 4.50
14.79 13.69 15.03 43.52 9.98 5.06 36.88 34.09 3.81 26.56 24.82 17.18 19.79 29.71
0.05 0.06 0.07 0.12 0.01 0.03 0.15 0.16 0.02 0.09 0.08 0.07 0.11 0.12
0.06 0.36 0.41 0.77 0.26 0.08 0.62 0.04 0.04 0.73 0.32 0.83 0.82 0.07
1.69 1.72 2.00 5.19 1.05 1.45 5.01 6.91 1.29 5.34 4.53 2.54 2.92 5.74
3.80 4.89 5.89 3.95 1.63 0.00 8.41 18.30 4.09 4.05 4.68 9.05 5.15 12.64
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
76.08 72.26 83.28 89.12 28.68 59.03 98.34 107.46 45.79 76.83 51.05 42.76 41.33 67.31
Cinnamomeae
Ci01-1 Ci01-2 Ci01-3 Ci01-4 Ci01-5 Ci01-6 Ci02 Ci03 Ci04 Ci05 Ci06-1 Ci06-2 Ci06-3
0.00 0.00 0.00 0.00 1.16 0.11 0.00 0.00 0.61 0.16 0.08 2.77 0.96
1.02 1.17 1.06 1.74 3.06 0.61 1.68 0.35 2.44 2.38 1.72 2.44 2.56
0.14 0.63 0.05 0.45 2.99 0.33 0.00 0.00 0.95 0.74 0.26 2.07 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.95 0.00 0.00 0.33 0.00 0.00 0.14
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.15 0.15 0.17 0.34 0.09 0.08 0.07 0.04 0.53 0.33 0.06 0.06 0.05
0.01 0.00 0.07 0.09 0.12 0.03 0.17 0.09 0.14 0.23 0.10 0.15 0.00
1.32 1.95 1.35 2.63 7.42 1.16 2.88 0.48 4.67 4.17 2.21 7.50 3.73
Caninae
Ca01 Ca02
0.00 0.00
8.86 0.00
4.32 20.30
0.00 0.00
1.46 0.19
0.07 0.00
0.00 0.00
0.76 0.00
8.32 17.40
0.01 0.04
0.06 0.01
0.40 0.81
6.50 2.79
0.00 0.00
0.00 0.00
30.78 41.52
Synstylae
Sy01 Sy02
0.00 0.00
0.00 0.00
0.00 0.00
0.52 0.00
2.36 1.56
0.04 0.03
0.00 0.00
2.16 0.30
31.45 22.28
0.12 0.05
0.52 0.10
1.93 1.01
9.80 5.16
0.00 0.00
0.00 0.00
48.90 30.49
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Gallicanae
D.W. = dry weight. a Flavonol glycosides: 1, quercetin 3-O-(200 -O-b-D-glucosyl)-b-D-galactoside; 2, quercetin 3-O-sophoroside; 3, kaempferol 3-O-sophoroside; 4, quercetin 3-O-b-D-galactoside; 5, quercetin 3-O-b-D-glucoside; 6, quercetin 3-O-(200 O-galloyl)-b-D-glucoside; 7, kaempferol 3-O-(200 -O-galloyl)-b-D-glucoside; 8, kaempferol 3-O-b-D-galactoside; 9, kaempferol 3-O-b-D-glucoside; 10, quercetin 3-O-b-D-xylopyranoside; 11, quercetin 3-O-a-L-arabinofuranoside; 12, kaempferol 3-O-a-L-arabinofuranoside; 13, kaempferol 3-O-a-D-rhamnoside; 14, quercetin 3-O-(200 -O-b-D-glucosyl)-b-D-xyloside; 15, kaempferol 3-O-(200 -O-b-D-glucosyl)-b-D-xyloside.
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Fig. 3. Distribution of Rosa species between factors Z1 and Z2 calculated by PCA of the content of flavonol glycosides. The x-axis corresponds to the first factor Z1, and the y-axis corresponds to the second factor Z2. The plots show the distribution of Rosa species in sections Gallicanae (Ga), Cinnamomeae (Ci), Caninae (Ca) and Synstylae (Sy). The proportion of Z1 and Z2 was 54.6% and 18.2%, respectively. Factor Z1 was positively affected by contribution of compounds 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13, and negatively affected by 14 and 15. Factor Z2 was mainly affected positively by compounds 1, 2 and 3, positively.
Cinnamomeae, the samples in section Gallicanae were widely distributed, with Type A in the first quadrant, Type B in the fourth quadrant and Type C in the second quadrant. The wide distribution of the samples in section Gallicanae indicated a wide variety of hybrid patterns for the samples. Type B may be further divided into two groups according to Z1 values (x-axis). The samples in section Caninae and Synstylae were distributed in Type C and Type B with lower Z1 values, respectively. The aglycones of flavonol glycosides determined in this report were quercetin and kaempferol, and the sugar moieties of these glycosides were classified into four groups according to number and type of sugars. As shown in Table 3, flavonol glycosides with a disaccharide composed of two hexoses, one hexose (including those with a galloyl group), one pentose and a disaccharide composed of one hexose and one pentose, were defined as S1, S2, S3 and S4, respectively. The ratios of the content of S1–S4 to the total contents of flavonol glycosides are shown in Table 3. Differences among the four sections were more distinctive than the individual glycosides shown in Table 2. The ratios of S4 were 0.00 in section Gallicanae, Caninae and Synstylae, and the ratios of S3 were 0.00 in section Cinnamomeae. In the section Synstylae, the ratios of S1 were also 0.00. PCA using the data in Table 3 was also performed, but the proportions of Z1 and Z2 were 83.7% and 13.1%, respectively. Factor Z1 was affected positively by contributions of components S2 (0.537) and S3 (0.514), and negatively by S1 (0.525) and S4 (0.415); factor Z2 was affected positively by contribution of component S4 (0.901). The PCA plot (not shown) indicated that the samples in this report could be divided into three groups according to Z1 values (x-axis), Type B (Z1 > 1.5), Type A + C (0.5 > Z1 > 0.6) and Type D (Z1 < 1.0). As discussed above, a notable characteristic of lower amounts of flavonol glycosides in the petals of Rosa species in section Cinnamomeae than in sections Gallicanae, Caninae and Synstylae was determined, and the average contents in section Cinnamomeae (3.19 ± 2.26 mg/g) was found to be less than one twentieth of that in Gallicanae (67.09 ± 23.31 mg/g). The species in section Cinnamomeae were closely interrelated in both results of PCA using the contents of 15 glycosides and the proportions of glycosides
grouped by type of sugar moiety. Another notable characteristic in the species in section Cinnamomeae was the presence of compounds 14 and 15, which were not detected in sections Gallicanae, Table 3 The proportion of groups of flavonol glycosides classified by sugar moiety. Section
a
Code No.
Flavonol glycoside groups as a percentage of the total flavonol glycoside content (%) S1 (1-3a)
S2 (4-9,13)
S3 (10-12)
S4 (14,15)
Gallicanae
Ga01-1 Ga01-2 Ga01-3 Ga02 Ga03 Ga04 Ga05 Ga06 Ga07 Ga08 Ga09 Ga10 Ga11 Ga12
45.71 42.58 43.89 0.31 0.45 79.36 0.46 0.38 70.26 0.42 2.64 0.16 0.19 0.00
51.93 54.45 53.13 92.86 94.94 17.99 93.66 93.00 26.80 91.57 87.70 91.79 90.49 91.19
2.37 2.96 2.98 6.82 4.60 2.64 5.88 6.62 2.95 8.02 9.66 8.04 9.32 8.81
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Cinnamomeae
Ci01-1 Ci01-2 Ci01-3 Ci01-4 Ci01-5 Ci01-6 Ci02 Ci03 Ci04 Ci05 Ci06-1 Ci06-2 Ci06-3
87.88 92.31 82.22 83.59 97.17 90.52 58.54 72.92 85.65 78.66 92.79 97.20 97.88
0.00 0.00 0.00 0.00 0.00 0.00 33.10 0.00 0.00 7.91 0.00 0.00 3.77
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
12.12 7.69 17.78 16.41 2.83 9.48 8.36 27.08 14.35 13.43 7.21 2.80 1.35
Caninae
Ca01 Ca02
42.85 48.87
55.62 49.06
1.53 2.07
0.00 0.00
Synstylae
Sy01 Sy02
0.00 0.00
94.74 96.20
5.26 3.80
0.00 0.00
Compound No.: see footnote of Table 2.
O. Sarangowa et al. / Phytochemistry 107 (2014) 61–68
Caninae, or Synstylae. In section Gallicanae, fourteen farm-raised and commercial samples were analyzed. The results of quantitative analysis and PCA indicated that the samples could be divided into three groups, because roses in section Gallicanae were complex hybrids of R. gallica, Rosa centifolia, and Rosa damascena, etc., and many horticultural roses are commercially available. Characteristics including contents of glycosides among the producing properties of these roses are influenced by the species used for breeding. 4. Concluding remarks In conclusion, the quantitative analysis of glycosides in the petals of Rosa species determined by UPLC provides a potent classification tool for sections Gallicanae, Cinnamomeae, Caninae and Synstylae.
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5.3. Extraction and isolation of flavonol glycosides Pulverized petals (348 g) collected in Hotan, Xiyumeigui farm, Xinjiang province in China (Ga01-1), were extracted five times with EtOH–H2O (8 l, 50:50, v/v) at room temperature for 24 h. After removal of the EtOH, the combined extracts were lyophilized. Then the resulting extract (150 g) was partitioned three times between MeOH:H2O:CHCl3 (1:1:2). The upper layer was evaporated to obtain crude material (135 g). The upper layer was suspended in H2O and was applied to a column of MCI gel CHP-20P (5 i.d. 47 cm). The column was eluted with a solvent system of H2O/MeOH/acetone to obtain fractions (Fraction 1–5). Fractions 2 (28 g) and 3 (12 g) were further purified by chromatography on Sephadex LH-20 and Wakosil 40 C18 columns to give compounds 1–13: compound 1 (120.0 mg), 2 (248.0 mg), 3 (423.0 mg), 4 (81.6 mg), 5 (4.63 mg), 6 (32.5 mg), 7 (9.95 mg), 8 (15.1 mg), 9 (264.2 mg), 10 (5.57 mg), 11 (20.9 mg), 12 (7.75 mg) and 13 (104.0 mg).
5. Experimental 5.1. General experimental procedures 1
H (500 MHz) and 13C NMR (125 MHz) spectra were measured with a JEOL a-500 spectrometer in CD3OD at 30 °C. Chemical shifts were determined using CD3OD (dH: 3.3 ppm, dC: 49.0 ppm) as the internal reference. APCI-TOF-MASS spectra were recorded on a JEOL JMS-T100LC spectrometer. Optical rotation was measured on a JASCO P-2200 polarimeter. MCI gel CHP-20 (Mitsubishi Chemical, Japan), Sephadex LH-20 (GE Healthcare, Sweden) and Wakosil 40 C18 (Wako Pure Chemical Industries, Ltd., Japan) were used for open column chromatography (CC). Ultra-performance liquid chromatography (UPLC) was performed using an Acquity UPLC system (Waters, USA) comprised of a binary solvent pump system, a sample manager, a column heater, a PDA detector, and an Acquity UPLC BEH C18 (2.1 i.d. 100 mm, 1.7 lm) column. All chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 5.2. Plant material Plants of R. gallica were not available from the wild, thus farmraised plants (Ga01-1–3) were collected in the Xinjiang province, China and commercially available hybrids of R. gallica were obtained from Yurigahara Lily Park, Sapporo (Ga03–07 and Ga10), and Chippubetsu Rose Garden, Chippubetsu, Hokkaido (Ga02, Ga09 and Ga11), Japan, respectively. Ga08 was the preserved sample of a hybrid of R. gallica collected at the Research Center for Medicinal Plant Resources National Institute of Biomedical Innovation, Nayoro, Hokkaido, Japan. Plants of R. rugosa growing in the wild were collected in Japan, Korea, and China, and wild plants of R. davurica were collected in Kamikawa, Hokkaido, Japan. Preservation plants belonging to section Cinnamomeae were collected at the Experimental Station for Medicinal Plant Studies Hokkaido University and Hokkaido (Ci04), Forestry Research Institute, Bibai, Hokkaido, (Ci02, Ci05 and Ci06-1), Japan. Two commercially available samples (Ci03 and Ci06-3) were obtained from Yurigahara Lily Park, Sapporo, Japan. Preservation samples of Rosa canica (Ca01) and Rosa multiflora (Sy01) were obtained from Hokkaido Forestry Research Institute, and two commercial samples (Ca02 and Sy02) were obtained from Yurigahara Lily Park, Sapporo, Japan. The species, collection reference, collection date, and facilities from which the samples are list below in Table 1. Fresh petals were separated and air-dried, and kept at room temperature in sealed package until used for experiments.
5.3.1. Quercetin 3-O-(200 -O-glucosyl)-b-D-galactoside (1) (Xiao et al., 2006) Brownish amorphous powder; [a]28 6.4° (c 3.0, MeOH); D Positive APCI-MS m/z 649.4 [M+Na]+. 5.3.2. Quercetin3-O-sophoroside (2) (Iwashina et al., 2005; Ross et al., 2005) Brownish amorphous powder; [a]28 D 23.1° (c 1.0, MeOH); Positive APCI-MS m/z 649.5 [M+Na]+. 5.3.3. Kaempferol3-O-sophoroside (3) (Schliemann et al., 2006; Ross et al., 2005) Yellow needles; [a]28 D 41.4° (c 1.0, MeOH); Positive APCI-MS m/z 633.5 [M+Na]+. 5.3.4. Quercetin3-O-b-D-galactoside (4, hyperoside) (Xiao et al., 2006; Zhu et al., 2013) Yellow needles; [a]28 D 9.1° (c 1.0, MeOH); Positive APCI-MS m/z 487.4 [M+Na]+. 5.3.5. Quercetin3-O-b-D-glucoside(5, isoquercitrin) Yellow needles; [a]28 D 5.0° (c 0.8, MeOH); Positive APCI-MS m/z 487.3 [M+Na]+. 5.3.6. Kaempferol3-O-b-D-galactoside (8, trifolin) Yellow needles; [a]28 D 15.7° (c 0.3, MeOH); Positive APCI-MS m/z 471.3 [M+Na]+. 5.3.7. Kaempferol3-O-b-D-glucoside (9, astragalin) (Xiao et al., 2006) Yellow needles; [a]28 D 11.4° (c 1.0, MeOH); Positive APCI-MS m/z 471.1 [M+Na]+. 5.3.8. Quercetin3-O-b-D-xylopyranoside (10, reynoutrin) (Zhu et al., 2013) Yellow amorphous powder; [a]28 D 17.8° (c 0.7, MeOH); Positive APCI-MS m/z 457.3 [M+Na]+. 5.3.9. Quercetin3-O-a-L-arabinofuranoside (11, avicularin) (Xiao et al., 2006; Park et al., 2012; Zhu et al., 2013) Brownish amorphous powder; [a]28 D 93.7° (c 1.0, MeOH); Positive APCI-MS m/z 457.3 [M+Na]+. 5.3.10. Kaempferol3-O-a-L-arabinofuranoside (12, juglanin) (Xiao et al., 2006; Zhu et al., 2013) Yellow needles; [a]28 D 121.1° (c 0.8, MeOH); Positive APCI-MS m/z 441.3 [M+Na]+.
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5.3.11. Kaempferol3-O-a-D-rhamnoside (13) (Nowak and Wolbis, 2002; Alaniya et al., 2012; Park et al., 2012) Yellow needles; [a]28 D 113.9° (c 0.4, MeOH); Positive APCI-MS m/z 455.2 [M+Na]+.
5.4. UPLC analysis Pulverized petals (1.00 g) of 31 samples used in this study, were individually extracted with EtOH:H2O (20 ml, 50:50, v/v) in an ultrasonic bath for 30 min. The supernatant was collected by centrifugation (1000g, 20 min), and was filtered through a 0.45 lm PTFE Millipore filter unit (Advantec, Japan), and then 1 ll of the filtrate was injected into the UPLC for analysis. The initial mobile phase (A) was CH3CN:H2O (2.5:97.5, v/v) containing 0.1% (v/v) HCO2H, and the final mobile phase (B) was CH3CN with 0.1% HCO2H. The gradient conditions were as follows: 0–4 min, 0% B; 26 min, 10% B; 45 min, 15% B; 51 min, 30% B; 54 min, 60% B; 55 min, 100% B. The flow rate was 0.2 ml/min. The detector wavelength range was set from 200 to 500 nm and the wavelength for quantitative determination was set at 350 nm.
5.5. Principal component analyses (PCA) PCA calculations were performed using IBM SPSSÒStatistics (Ver. 21.0). The contents of 15 flavonol glycosides and the percent ratio of four groups of flavonol glycosides classified by sugar moiety were used for original data.
Acknowledgments We thank Dr. Toshiro Shibata of the Research Center for Medicinal Plant Resources (Nayoro, Japan), National Institute of Biomedical Innovation, and Mr. Naotoshi Yoshida of the Hokkaido University, Medicinal Plant Garden, for kindly supplying the sample of Rosa spp. We also thank Mr. Guo Yong-Lai of the Pingying Rose Research Institute of China, and Prof. Mariya Sakim of the Xinjiang Medical University of China, for kindly supplying samples of Mei-gui.
References Alaniya, M.D., Kavtaradze, N.Sh., Skhirtladze, A.V., Sutiashvili, M.G., Kemertelidze, E.P., 2012. Flavonoid glycosides from flowers of Sisymbrium officinale and Diplotaxis muralis growing in Georgia. Chem. Nat. Compd. 48, 315–316. Gault, S.M., Synge, P.M., 1971. The Dictionary of Roses in Color. Ebury Press, Hague, p. 269. Grossi, C., Roymond, O., Jay, M., 1998. Flavonoid and enzyme polymorphisms and taxonomic organization of Rosa sections: Carolinae, Cinnamomeae, Pimpinellifoliae and Synstylae. Biochem. Syst. Ecol. 26, 857–871. Gu, G.Z., Kenneth, R.R., 2003. Rosa Linnaeus sp. Flora of China, vol. 9. Science Press (Beijing) and Missouri Botanical Garden Press. pp. 339–381. Iwashina, T., Kitajima, J., Shiuchi, T., Itou, Y., 2005. Chalcones and other flavonoids from Asaram sensu lato (Aristolochiaceae). Biochem. Syst. Ecol. 33, 571–584. Lin, J.H., Lin, Y.T., 1999. Flavonoids from the leaves of Loranthuskaoi (Chao) Kiu. J. Food Drug Anal. 7, 185–190. Mikanagi, Y., Saito, N., Yokoi, M., 1990. Flavonoid distribution in the flowers of genus Rosa sections Synstylae, Cinnamomeae, and Pimpinellifoliae. Flavonoids in Biology and Medicine III, Current Issues in Flavonoid Research, Singapore. pp. 89–96. Mikanagi, Y., Yokoi, M., Ueda, Y., Saito, N., 1995. Flower flavonol and anthocyanin distribution in subgenus Rosa. Biochem. Syst. Ecol. 23, 183–200. Muhammet, A.A., 2002. MizaniTib (1871). Xinjiang Science Healthy Publication, Xinjiang (China), p411, p421. Nowak, S., Wolbis, M., 2002. Flavonoids from some species of genus Scopolia Jacq. Acta Pol. Pharm. 59, 275–280. Park, B.J., Matsuta, T., Kanazawa, T., Park, C.H., Chang, K.J., Onjo, M., 2012. Phenolic compounds from the leaves of Psidium guajava II. Quercetin and its glycosides. Chem. Nat. Compd. 48, 477–479. Ross, S.A., Elsohly, M.A., Sultana, G.N.N., Mehmedic, Z., Hossain, C.F., Chandra, S., 2005. Flavonoid glycosides and cannabinoids from the pollen of Cannabis sativa L.. Phytochem. Anal. 16, 45–48. Sarangowa, O., Ishii, K., Park, B.J., Matsuta, T., Nishizawa, M., Kanazawa, T., Funaki, M., Yamagishi, T., 2010. Botanical origin of Mei-gui Hua (petal of a Rosa species). J. Nat. Med. 64, 409–416. Sarangowa, O., Yuki, T., Kanazawa, T., Nishizawa, M., Yamagishi, T., 2013. Two galloylated flavonoids as antioxidants in Rosa gallica petal. Chem. Nat. Compd. 49, 940–942. Schliemann, W., Schneider, B., Wray, V., Schmidt, J., Nimtz, M., Porzel, A., Bohm, H., 2006. Flavonols and an indole alkaloid skeleton bearing identical acylated glycosidic groups from yellow petals of Papavernudicaule. Phytochemistry 67, 191–201. Tanaka, N., Murakami, T., Saiki, Y., Chen, C.M., Gomez, D., 1981. Chemical and chemotaxonomical studies of Ferns. XXXVII. Chemical studies on the constituents of Costa Rican ferns (2). Chem. Pharm. Bull. 29, 3455–3463. Xiao, Z.P., Wu, H.K., Wu, T., Shi, H., Hang, B., Aisa, H.A., 2006. Kaempferol and quercetin flavonoids from Rosa rugosa. Chem. Nat. Compd. 42, 736–737. Zhu, Y., Liu, Y., Zhan, Y., Liu, L., Xu, Y., Xu, T., Liu, T., 2013. Preparative isolation and purification of five flavonoid glycosides and one benzophenone galloyl glycoside from Psidium guajava by high-speed counter-current chromatography (HSCCC). Molecules 18, 15648–15661.
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Flavonol glycosides in the petal of Rosa species as chemotaxonomic markers Ochir Sarangowa a,1, Tsutomu Kanazawa a, Makoto Nishizawa b, Takao Myoda b, Changxi Bai c, Takashi Yamagishi a,⇑ a b c
Kitami Institute of Technology, 165 Koen-cho, Kitami 090-8507, Japan Faculty of Bio-Industry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan Academy of Mongolian Medicine, Inner Mongolia Medical University, Hohhot 010110, China
a r t i c l e
i n f o
Article history: Received 22 March 2014 Received in revised form 30 July 2014 Available online 11 September 2014 Keywords: Rosa species Rosa gallica Rosa rugosa Rosaceae Quercetin glycoside Kaempferol glycoside Chemotaxonomy
a b s t r a c t Thirteen flavonol glycosides were isolated from the petals of Rosa species belonging to the section Gallicanae, and their structures were identified from their spectroscopic data. These flavonol glycosides, along with two flavonol glycosides isolated from Rosa rugosa, in the petals of 31 Rosa species belonging to sections Gallicanae, Cinnamomeae, Caninae, and Synstylae were quantitatively analyzed by UPLC. The results indicated that the species belonging to these sections could be classified into four types (Type A, B, C and D) based on the pattern of flavonol glycoside contents, whereas the R. rugosa flavonol glycosides were detected only in section Cinnamomeae. A principal components analysis (PCA) calculated from the 15 flavonol glycosides contained in these samples supported the presence of four types. The distribution of the species in Type D (a group of Cinnamomeae) was shown to reflect close interrelationships, but species in Type B (one group of Gallicanae) could be subdivided into two groups, one of which contained species in section Synstylae. Moreover, the flavonol glycosides were grouped by sugar moieties: a disaccharide composed of two hexoses (S1), a hexose (S2), including a hexose with galloyl group, a pentose (S3), and a disaccharide composed of a hexose and a pentose (S4). The ratios of the amounts of S1–S4 to total flavonol glycoside content indicated that differences among the four sections were more distinctive than the amounts of the 15 flavonol glycosides. The 31 samples were divided into Type B, composed of one type of Gallicanae and Synstylae, Type A + C, composed of another type of Gallicanae and Caninae, and Type D, composed of Cinnamomeae. The R. rugosa flavonol glycosides were shown to be important chemotaxonomic markers for the classification of species in Cinnamomeae, and this method of using flavonol glycosides as chemotaxonomic markers could be useful for the identification of Rosa species belonging to sections Gallicanae, Cinnamomeae, Caninae, and Synstylae. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Petals and blossoms of roses have been used for medicines and as an ingredient of herbal tea in eastern Asia. As the original rose plants differ depending on country, the aim herein was to characterize original plants by phytochemical methods. In Japan, Rosa rugosa, a wild rose distributed mainly in the northern islands, has been used for an herbal tea (Gault and Synge, 1971). In China, roses used for medicines and in herbal teas ⇑ Corresponding author. Address: Applied Research Center, Kitami Institute of Technology, 165 Koen-cho, Kitami 090-8507, Japan. Tel.: +81 157 26 9462; fax: +81 157 26 9461. E-mail address: [email protected] (T. Yamagishi). 1 Present address: Academy of Mongolian Medicine, Inner Mongolia Medical University, Hohhot 010110, China. http://dx.doi.org/10.1016/j.phytochem.2014.08.013 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
are called ‘‘Mei-gui Hua’’, but many kinds of Mei-gui Hua prepared from different plant species are commercially available (Gu and Kenneth, 2003). In the Xinjiang province, the westernmost part of China, Mei-gui Hua made from the blossoms or petals of roses cultivated there are called ‘‘Kizil gul’’, and they have been used in herbal teas for the prevention and treatment of diabetes (Muhammet, 2002). In a previous paper, morphological, phylogenic and phytochemical studies established that the plant species of Mei-gui Hua cultivated in the Xinjiang province must be Rosa gallica or its hybrids, whereas those cultivated in the northeastern part of China are hybrids of R. rugosa. In phytochemical studies, the 3-O-b-D-glucosides of quercetin and kaempferol were not detected in petals of wild R. rugosa collected in Japan and Korea. However, the 3-O(200 -O-b-D-glucosyl)-b-D-xylosides of quercetin and kaempferol
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were detected in the petals of R. rugosa, but were not detected in Mei-gui Hua cultivated in the Xinjiang province. There have also been some reports on the characterization of sections of subgenus Rosa using flavonol glycosides, thus the content of flavonol glycosides may be useful to characterize plant species belonging to sections of subgenus Rosa for medicinal use (Sarangowa et al., 2010). Therefore, the content of 15 flavonol glycosides (Fig. 1) are quantitatively determined in the petals of roses belong to sections Gallicanae and Cinnamomeae. In this paper, the usefulness of flavonol glycosides for chemotaxonomy of these sections belonging to subgenus Rosa are reported.
hexose, and the spectra of 12 and 13 showed the presence of a pentose. Comparing the 1H and 13C NMR data and MS data of these compounds with literature values (Nowak and Wolbis, 2002; Alaniya et al., 2012; Park et al., 2012), the structures of 8, 12 and 13 were characterized as kaempferol 3-O-b-D-galactoside (8), kaempferol 3-O-a-L-arabinofuranoside (12), and kaempferol 3-Oa-D-rhamnoside (13). Quercetin 3-O-(200 -O-galloyl)-b-D-glucoside (6) and kaempferol 3-O-(200 -O-galloyl)-b-D-glucoside (7) were also isolated from R. gallica as detailed in our previous report (Sarangowa et al., 2013) (Fig. 1).
2. Results
2.2. Quantitative determination of flavonol glycosides in sections Gallicanae, Cinnamomeae, Caninae and Synstylae
2.1. Isolation of flavonol glycosides from R. gallica The 50% EtOH extracts of the petals of a hybrid of R. gallica collected in the Xinjiang province in China were continuously partitioned with a H2O–MeOH–CHCl3 system, and the upper layer was fractionated by column chromatography on MCI gel CHP-20P followed by chromatography on Sephadex LH-20. The fractions containing flavonol glycosides were further purified using a reversed phase gel (Wakosil 40 C18) to give compounds 1–13. Compounds 1, 2, 3, 5 and 9 were isolated as the major flavonol glycosides in the petals of R. gallica, and were identified as quercetin 3-O-(200 O-b-D-glucosyl)-b-D-galactoside (1), quercetin 3-O-sophoroside (2), kaempferol 3-O-sophoroside (3), quercetin 3-O-b-D-glucoside (5) and kaempferol 3-O-b-D-glucoside (9), respectively, as previously described (Sarangowa et al., 2010) and by comparison of their 1H and 13C NMR data and MS data with literature values (Iwashina et al., 2005; Schliemann et al., 2006; Ross et al., 2005). The 1H NMR spectra of 4, 10 and 11 indicated the presence of quercetin as an aglycone. The 13C NMR spectrum of compound 4 showed the presence of a hexose, and the spectra of 10 and 11 showed the presence of a pentose. Comparing the 1H and 13C NMR data and MS data with literature data (Lin and Lin, 1999; Nowak and Wolbis, 2002; Park et al., 2012; Tanaka et al., 1981; Zhu et al., 2013), the structures of 4, 10 and 11 were identified as quercetin 3-O-b-D-galactoside (4), quercetin 3-O-b-D-xylopyranoside (10) and quercetin 3-O-a-L-arabinofuranoside (11), respectively. The positions of the sugar moiety on quercetin were confirmed by analysis of HMBC spectra. The C1–H of the sugar moiety gave a cross peak with the C3 position of quercetin. The 1H NMR spectra of compounds 8, 12 and 13 indicated presence of kaempferol as the aglycone (Xiao et al., 2006; Zhu et al., 2013). The 13C NMR spectrum of 8 showed the presence of a
glycosides of quercetin 1: R= 3-O-(2"-O-β-D-glucosyl)-β-D-galctoside 2: R= 3-O-sophoroside 4: R= 3-O-β-D-galctoside 5: R= 3-O-β-D-glucoside 6: R=3-O-(2"-O-galloyl)-β-D-glucoside 10: R= 3-O-β-D-xylopyranoside 11: R= 3-O-α-L-arabinofuranoside 14: R= 3-O-(2"-O-β-D-glucosyl)-β -D-xyloside
A method using ultra-performance liquid chromatography (UPLC) equipped with a photodiode array (PDA) detector was optimized for quantitative determination of flavonol glycosides in the 50% EtOH extract of the petal of 31 Rosa species (Table 1). The monitoring wavelength was set at 350 nm, which was the absorption maximum for glycosides of quercetin and kaempferol. Chromatography peaks of quercetin and kaempferol glycosides were identified from their retention times and UV spectra monitored by PDA detector. The content of flavonol glycosides was quantitatively analyzed by UPLC. Typical chromatograms of hybrids of R. gallica and R. rugosa are shown in Fig. 2. Seven glycosides of quercetin (1, 2, 4, 5, 6, 10 and 11) and six glycosides of kaempferol (3, 7, 8, 9, 12 and 13) were detected in the chromatogram of the hybrid of R. gallica. However, in the chromatogram of R. rugosa, 3-O-(200 -O-b-D-glycosyl)b-D-xylosides of quercetin (14) and kaempferol (15) were detected along with 2 and 3, while other glycosides were not detected. The content of fifteen glycosides of quercetin and kaempferol are shown in Table 2. Fifteen flavonol glycosides were identified in this experiment; however, all of the flavonol glycosides were not detected in all samples. The total content of these glycosides was highest in the petals of Rosa species in section Gallicanae (28.68–119.10 mg/g), followed by section Caninae (30.78 and 41.52 mg/g) and section Synstylae (30.49 and 48.90 mg/g). The glycoside content in the petals of Rosa species in Section Cinnamomeae was lower than the other sections (0.48–7.5 mg/g). Flavonol glycosides including a hexose (4, 5, 8, 9 and 13) and their galloyl derivatives (6 and 7), and those with a pentose (10, 11 and 12) were detected only in section Gallicanae, except for Rosa acicularis (Ci05), Rosa davurica (Ci08) and R. rugosa Rosa rubrifoia
glycosides of kaempferol 3: R= 3-O-sophoroside 7: R= 3-O-(2"-O-galloyl)-β-D-glucoside 8: R= 3-O-β-D-galctoside 9: R= 3-O-β-D-glucoside 12: R= 3-O-α-L-arabinofuranoside 13: R= 3-O-α-D-rhamnoside 15: R= 3-O-(2"-O-β-D-glucosyl)-β-D-xyloside
Fig. 1. Glycosides of quercetin and kaempferol.
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O. Sarangowa et al. / Phytochemistry 107 (2014) 61–68 Table 1 List of samples. Sample code
Species
Collection reference
Date collected
Section Gallicanae Ga01-1 Ga01-2 Ga01-3 Ga02 Ga03 Ga04 Ga05 Ga06 Ga07 Ga08 Ga09 Ga10 Ga11 Ga12
R. R. R. R. R. R. R. R. R. R. R. R. R. R.
gallica (hybrid) gallica (hybrid) gallica (hybrid) gallica var. officinalis Ser. gallica ‘Complicate’ gallica ‘Alika’ gallica ‘Charles de Mills’ gallica ‘OeilletFlamand’ gallica ‘James Mason’ gallica ‘Francofurtana’ centifolia var. sancta (Richard) Zabel damascena ‘Bifera’ damascena ‘Perpetual’ damascena ‘Jacques Cartier’
Farm-raised Farm-raised Farm-raised Commercial Commercial Commercial Commercial Commercial Commercial Preservation Commercial Commercial Commercial Commercial
02/05/2007 01/05/2007 01/05/2007 06/07/2010 25/06/2010 25/06/2010 25/06/2010 25/06/2010 25/06/2010 28/09/2010 06/07/2011 02/07/2012 02/07/2012 13/08/2009
Section Cinnamomeae Ci01-1 Ci01-2 Ci01-3 Ci01-4 Ci01-5 Ci01-6 Ci02 Ci03 Ci04 Ci05 Ci06-1 Ci06-2 Ci06-3
R. R. R. R. R. R. R. R. R. R. R. R. R.
rugosa Thunb. rugosa Thunb. rugosa Thunb. rugosa Thunb. rugosa Thunb. rugosa Thunb. rugosa R. rubrifolia rugosa var. rubra Rehder maikwai Hara acicularis Lindl. davurica Pall.var. alpestris (Nakai) Kitag. davurica Pall.var. alpestris (Nakai) Kitag. davurica Pall.var. alpestris (Nakai) Kitag.
Wild Wild Wild Wild Wild Wild Preservation Commercial Preservation Preservation Preservation Wild Commercial
26/08/2007 05/05/2009 06/07/2010 06/07/2010 06/07/2010 06/07/2010 02/07/2011 25/06/2010 06/07/2010 06/07/2010 18/06/2011 29/06/2007 25/06/2010
Section Caninae Ca01 Ca02
R. rubrifolia Vill. (R. glauca Pourr.) R. canina L.
Preservation Commercial
02/07/2011 25/06/2010
Section Synstylae Sy01 Sy02
R. multiflora Thunb. R. gentileana Léveillé & Vaniot
Preservation Commercial
02/07/2011 25/06/2010
Ga01-1: Hotan, Xiyumeigui farm, Xinjiang province, China. Ga01-2: Niya, Xinjiang Province, China. Ga01-3: Keriye, Xinjiang Province, China. Ga02, Ga09 and Ga12: Chippubetsu Rose Garden, Chippubetsu, Hokkaido, Japan. Ga03, Ga04, Ga05, Ga06, Ga07, Ga10, Ga11, Ci03, Ci06-3 Ca02 and Sy02: Yurigahara Lily Park, Sapporo, Japan. Ga08: The Research Center for Medicinal Plant Resources National Institute of Biomedical Innovation, Nayoro, Hokkaido, Japan. Ci01-1: Komuke Natural Flower Garden, Monbetsu, Hokkaido, Japan. Ci01-2: Sado Island, Niigata, Japan. Ci01-3: Yangyang, Gangwon-do, Korea. Ci01-4: HwasongSongsan, Gangwon-do, Korea. Ci01-5: Pingying, Shandong Province, China. Ci02, Ci05, Ci06-1, Ca01 and Sy01: Hokkaido Forestry Research Institute, Bibai, Hokkaido, Japan. Ci04: Experimental Station for Medicinal Plant Studies, Hokkaido University, Sapporo, Japan. Ci06-2: Kamikawa, Hokkaido, Japan. All samples are kept in The Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation, Nayoro, Hokkaido, Japan (Yamagishi crude drag library, code No. Ga01-1, Ga01-2, Ga01-3, Ga02, Ga03, Ga04, Ga05, Ga06, Ga07, Ga08, Ga09, Ga10, Ga11, Ga12, Ci01-1, Ci01-2, Ci01-3, Ci01-4, Ci01-5, Ci01-6, Ci06-1, Ci06-2, Ci06-3, Ca01, Ca02, Sy01, Sy02 Ci02, Ci03, Ci04, Ci05).
(Ci02). On the other hand, the Rosa species in section Cinnamomeae mainly contained glycosides with a disaccharide (1, 2, 3, 14 and 15). Sophorosides of quercetin and/or kaempferol (2 and 3, respectively) were detected in almost all of the samples, and two patterns involving the presence and content of these glycosides along with the content of 1 were recognized. Samples belong to pattern 1 contained relatively large amount of 1, 2 and 3, and those belonging to pattern 2 contained lower amounts of these compounds (or were not detected). Compounds 14 and 15 were detected only in section Cinnamomeae. 3. Discussion
collected in the Xinjiang province, China. In the present study, samples of Rosa plants were collected at the same location for analysis. As reported previously, the roses used as ‘‘Mei-gui’’ or ‘‘Kizil gul’’ in Xinjiang province are not R. rugosa, but are R. gallica and its hybrids. In this paper, glucosides 4, 9, 11 and 12 were not detected in 6 samples of R. rugosa collected in Japan, Korea and China, and 1, 3, 4, 9, 11 and 12 were detected in all of the samples collected in Xinjiang province. Therefore, the plants collected by Xiao et al. (2006) were supposed to be hybrids of R. gallica. In this study, additional glycosides of quercetin and kaempferol including 2, 5, 8, 10 and 13 were isolated for the first time from hybrids of R. gallica.
3.1. Flavonol glycosides in R. gallica
3.2. Flavonol glycosides as chemotaxonomic markers
Xiao et al. (2006) reported the isolation of glycosides of kaempferol and quercetin such as 1, 3, 4, 9, 11 and 12 from ‘‘R. rugosa’’
Mikanagi et al. (1990, 1995) reported that quercetin 3O-glucoside (5) and kaempferol 3-O-glucoside (9) were the major
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O. Sarangowa et al. / Phytochemistry 107 (2014) 61–68
Fig. 2. UPLC Chromatograms of the 50% EtOH extract of the petal. a: R. gallica b: R. rugosa. 1, quercetin 3-O-(200 -O-b-D-glucosyl)-b-D-galactoside; 2, quercetin 3-O-sophoroside; 3, kaempferol 3-O-sophoroside; 4, quercetin 3-O-b-D-galactoside; 5, quercetin 3-O-b-D-glucoside; 6, quercetin 3-O-(200 -O-galloyl)-b-D-glucoside; 7, kaempferol 3-O-(200 -Ogalloyl)-b-D-glucoside; 8, kaempferol 3-O-b-D-galactoside; 9, kaempferol 3-O-b-D-glucoside; 10, quercetin 3-O-b-D-xylopyranoside; 11, quercetin 3-O-a-L-arabinofuranoside; 12, kaempferol 3-O-a-L-arabinofuranoside; 13, kaempferol 3-O-a-D-rhamnoside; 14, quercetin3-O-(200 -O-b-D-glucosyl)-b-D-xyloside; 15, kaempferol 3-O-(200 -O-b-D-glucosyl)b-D-xyloside.
flavonoid glycosides in section Gallicanae. In this study, flavonol glycosides in the hybrids of R. gallica were analyzed, and compounds 5 and 9 were detected in all samples. However, the results indicated that samples in this section could be divided into three types according to the content level of these flavonol glycosides. Type A was composed of three samples (Ga01-1–Ga01-3) collected in the Xinjiang province in China, containing 1, 2, 3, 4 and 13 in high levels along with 5 and 9. Type B was composed of nine samples (Ga02–03, Ga05–06 and Ga08–12), containing 5 and 9 as the major flavonol glycosides, but the content of 2 and 3 was low, and 1 was not detected. Type C was composed of two samples (Ga04 and Ga07), containing 1, 2 and 3 in high content, and 5 and 9 were in low content. Contrary to Mikanagi’s reports, these results showed that compounds 5 and 9 were not necessarily the major flavonol glycosides, which indicated that these samples were hybrids of R. gallica. In fourteen samples of Gallicanae, 9 was detected in all of the samples, but the contents of 5 was low in some samples. These results here indicated that a notable feature of Rosa species in section Cinnamomeae was their low contents of flavonol glycosides (0.48–7.50 mg/g). The average total contents of flavonol glycosides (3.19 ± 2.26 mg/g) was more than twenty fold lower for Cinnamomeae than for Gallicanae (67.09 ± 23.31 mg/g). Mikanagi et al. (1995) and Grossi et al. (1998) reported that quercetin 3-O-sophoroside (2) and kaempferol 3-O-sophoroside (3) were characteristic markers of the section Cinnamomeae. In the results here, compound 2 was detected in all Cinnamomeae
samples, but compound 3 was not detected in all samples, and some samples contained compound 1. On the other hand, these results indicated that the more characteristic flavonol glycosides were the 3-O-(20 -O-b-D-glycosyl)-b-D-xyloside of quercetin (14) and kaempferol (15). This characteristic feature of section Cinnamomeae was defined as Type D, as there were no samples in Gallicanae, Caninae, or Synstylae that contained 14 and 15. Therefore, 14 and 15 could be important chemotaxonomic markers of section Cinnamomeae. Grossi et al. (1998) have also reported that section Synstylae was well defined by the presence of kaempferol 3-O-glucoside (9) and kaempferol 3-O-rhamnoside (13). The results here were consistent with their report, as compound 9 was the major flavonol glycoside followed by 13 identified in this subspecies. There was no characteristic feature of flavonol glycoside content in the samples in section Caninae and Synstylae, which was similar to those of Type B and Type C of section Gallicanae, respectively. The total contents of flavonol glycosides in these three sections were higher than those in section Cinnamomeae. Principal component analysis (PCA) of the content of fifteen glycosides of quercetin and kaempferol in 31 Rosa species was performed using SPSS Statistics software. As shown in Fig. 3, the distribution plots were divided into five groups. In the third quadrant, thirteen species in section Cinnamomeae (Type D) were closely distributed. This close distribution of the samples in Cinnamomeae must be due to the samples in section Cinnamomeae all being wild or preservation plants. In contrast to
Table 2 Contents of flavonol glycosides in the petals of Rosa species. Section
Code No.
Content of flavonol glycosidesa (mg/g D.W.)
Total
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Ga01-1 Ga01-2 Ga01-3 Ga02 Ga03 Ga04 Ga05 Ga06 Ga07 Ga08 Ga09 Ga10 Ga11 Ga12
13.57 11.02 13.56 0.00 0.00 13.72 0.00 0.00 11.82 0.00 0.00 0.00 0.00 0.00
13.14 11.81 14.08 0.03 0.03 12.17 0.23 0.29 8.79 0.07 0.12 0.06 0.08 0.00
8.07 7.95 8.91 0.25 0.10 20.95 0.22 0.12 11.56 0.25 1.23 0.01 0.00 0.00
6.57 6.18 7.21 12.41 4.52 1.38 17.89 23.20 1.12 16.15 4.05 3.97 3.86 6.44
11.37 11.31 12.25 10.96 9.95 1.79 22.42 10.49 1.23 19.15 8.06 3.89 5.69 7.91
0.05 0.04 0.05 0.11 0.10 0.02 0.04 0.12 0.02 0.04 0.01 0.09 0.06 0.12
0.06 0.07 0.09 0.05 0.01 0.02 0.11 0.12 0.00 0.10 0.02 0.03 0.02 0.07
2.87 3.18 3.72 11.76 1.03 2.35 6.36 13.63 2.00 4.30 3.12 5.05 2.82 4.50
14.79 13.69 15.03 43.52 9.98 5.06 36.88 34.09 3.81 26.56 24.82 17.18 19.79 29.71
0.05 0.06 0.07 0.12 0.01 0.03 0.15 0.16 0.02 0.09 0.08 0.07 0.11 0.12
0.06 0.36 0.41 0.77 0.26 0.08 0.62 0.04 0.04 0.73 0.32 0.83 0.82 0.07
1.69 1.72 2.00 5.19 1.05 1.45 5.01 6.91 1.29 5.34 4.53 2.54 2.92 5.74
3.80 4.89 5.89 3.95 1.63 0.00 8.41 18.30 4.09 4.05 4.68 9.05 5.15 12.64
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
76.08 72.26 83.28 89.12 28.68 59.03 98.34 107.46 45.79 76.83 51.05 42.76 41.33 67.31
Cinnamomeae
Ci01-1 Ci01-2 Ci01-3 Ci01-4 Ci01-5 Ci01-6 Ci02 Ci03 Ci04 Ci05 Ci06-1 Ci06-2 Ci06-3
0.00 0.00 0.00 0.00 1.16 0.11 0.00 0.00 0.61 0.16 0.08 2.77 0.96
1.02 1.17 1.06 1.74 3.06 0.61 1.68 0.35 2.44 2.38 1.72 2.44 2.56
0.14 0.63 0.05 0.45 2.99 0.33 0.00 0.00 0.95 0.74 0.26 2.07 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.95 0.00 0.00 0.33 0.00 0.00 0.14
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.15 0.15 0.17 0.34 0.09 0.08 0.07 0.04 0.53 0.33 0.06 0.06 0.05
0.01 0.00 0.07 0.09 0.12 0.03 0.17 0.09 0.14 0.23 0.10 0.15 0.00
1.32 1.95 1.35 2.63 7.42 1.16 2.88 0.48 4.67 4.17 2.21 7.50 3.73
Caninae
Ca01 Ca02
0.00 0.00
8.86 0.00
4.32 20.30
0.00 0.00
1.46 0.19
0.07 0.00
0.00 0.00
0.76 0.00
8.32 17.40
0.01 0.04
0.06 0.01
0.40 0.81
6.50 2.79
0.00 0.00
0.00 0.00
30.78 41.52
Synstylae
Sy01 Sy02
0.00 0.00
0.00 0.00
0.00 0.00
0.52 0.00
2.36 1.56
0.04 0.03
0.00 0.00
2.16 0.30
31.45 22.28
0.12 0.05
0.52 0.10
1.93 1.01
9.80 5.16
0.00 0.00
0.00 0.00
48.90 30.49
O. Sarangowa et al. / Phytochemistry 107 (2014) 61–68
Gallicanae
D.W. = dry weight. a Flavonol glycosides: 1, quercetin 3-O-(200 -O-b-D-glucosyl)-b-D-galactoside; 2, quercetin 3-O-sophoroside; 3, kaempferol 3-O-sophoroside; 4, quercetin 3-O-b-D-galactoside; 5, quercetin 3-O-b-D-glucoside; 6, quercetin 3-O-(200 O-galloyl)-b-D-glucoside; 7, kaempferol 3-O-(200 -O-galloyl)-b-D-glucoside; 8, kaempferol 3-O-b-D-galactoside; 9, kaempferol 3-O-b-D-glucoside; 10, quercetin 3-O-b-D-xylopyranoside; 11, quercetin 3-O-a-L-arabinofuranoside; 12, kaempferol 3-O-a-L-arabinofuranoside; 13, kaempferol 3-O-a-D-rhamnoside; 14, quercetin 3-O-(200 -O-b-D-glucosyl)-b-D-xyloside; 15, kaempferol 3-O-(200 -O-b-D-glucosyl)-b-D-xyloside.
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O. Sarangowa et al. / Phytochemistry 107 (2014) 61–68
Fig. 3. Distribution of Rosa species between factors Z1 and Z2 calculated by PCA of the content of flavonol glycosides. The x-axis corresponds to the first factor Z1, and the y-axis corresponds to the second factor Z2. The plots show the distribution of Rosa species in sections Gallicanae (Ga), Cinnamomeae (Ci), Caninae (Ca) and Synstylae (Sy). The proportion of Z1 and Z2 was 54.6% and 18.2%, respectively. Factor Z1 was positively affected by contribution of compounds 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13, and negatively affected by 14 and 15. Factor Z2 was mainly affected positively by compounds 1, 2 and 3, positively.
Cinnamomeae, the samples in section Gallicanae were widely distributed, with Type A in the first quadrant, Type B in the fourth quadrant and Type C in the second quadrant. The wide distribution of the samples in section Gallicanae indicated a wide variety of hybrid patterns for the samples. Type B may be further divided into two groups according to Z1 values (x-axis). The samples in section Caninae and Synstylae were distributed in Type C and Type B with lower Z1 values, respectively. The aglycones of flavonol glycosides determined in this report were quercetin and kaempferol, and the sugar moieties of these glycosides were classified into four groups according to number and type of sugars. As shown in Table 3, flavonol glycosides with a disaccharide composed of two hexoses, one hexose (including those with a galloyl group), one pentose and a disaccharide composed of one hexose and one pentose, were defined as S1, S2, S3 and S4, respectively. The ratios of the content of S1–S4 to the total contents of flavonol glycosides are shown in Table 3. Differences among the four sections were more distinctive than the individual glycosides shown in Table 2. The ratios of S4 were 0.00 in section Gallicanae, Caninae and Synstylae, and the ratios of S3 were 0.00 in section Cinnamomeae. In the section Synstylae, the ratios of S1 were also 0.00. PCA using the data in Table 3 was also performed, but the proportions of Z1 and Z2 were 83.7% and 13.1%, respectively. Factor Z1 was affected positively by contributions of components S2 (0.537) and S3 (0.514), and negatively by S1 (0.525) and S4 (0.415); factor Z2 was affected positively by contribution of component S4 (0.901). The PCA plot (not shown) indicated that the samples in this report could be divided into three groups according to Z1 values (x-axis), Type B (Z1 > 1.5), Type A + C (0.5 > Z1 > 0.6) and Type D (Z1 < 1.0). As discussed above, a notable characteristic of lower amounts of flavonol glycosides in the petals of Rosa species in section Cinnamomeae than in sections Gallicanae, Caninae and Synstylae was determined, and the average contents in section Cinnamomeae (3.19 ± 2.26 mg/g) was found to be less than one twentieth of that in Gallicanae (67.09 ± 23.31 mg/g). The species in section Cinnamomeae were closely interrelated in both results of PCA using the contents of 15 glycosides and the proportions of glycosides
grouped by type of sugar moiety. Another notable characteristic in the species in section Cinnamomeae was the presence of compounds 14 and 15, which were not detected in sections Gallicanae, Table 3 The proportion of groups of flavonol glycosides classified by sugar moiety. Section
a
Code No.
Flavonol glycoside groups as a percentage of the total flavonol glycoside content (%) S1 (1-3a)
S2 (4-9,13)
S3 (10-12)
S4 (14,15)
Gallicanae
Ga01-1 Ga01-2 Ga01-3 Ga02 Ga03 Ga04 Ga05 Ga06 Ga07 Ga08 Ga09 Ga10 Ga11 Ga12
45.71 42.58 43.89 0.31 0.45 79.36 0.46 0.38 70.26 0.42 2.64 0.16 0.19 0.00
51.93 54.45 53.13 92.86 94.94 17.99 93.66 93.00 26.80 91.57 87.70 91.79 90.49 91.19
2.37 2.96 2.98 6.82 4.60 2.64 5.88 6.62 2.95 8.02 9.66 8.04 9.32 8.81
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Cinnamomeae
Ci01-1 Ci01-2 Ci01-3 Ci01-4 Ci01-5 Ci01-6 Ci02 Ci03 Ci04 Ci05 Ci06-1 Ci06-2 Ci06-3
87.88 92.31 82.22 83.59 97.17 90.52 58.54 72.92 85.65 78.66 92.79 97.20 97.88
0.00 0.00 0.00 0.00 0.00 0.00 33.10 0.00 0.00 7.91 0.00 0.00 3.77
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
12.12 7.69 17.78 16.41 2.83 9.48 8.36 27.08 14.35 13.43 7.21 2.80 1.35
Caninae
Ca01 Ca02
42.85 48.87
55.62 49.06
1.53 2.07
0.00 0.00
Synstylae
Sy01 Sy02
0.00 0.00
94.74 96.20
5.26 3.80
0.00 0.00
Compound No.: see footnote of Table 2.
O. Sarangowa et al. / Phytochemistry 107 (2014) 61–68
Caninae, or Synstylae. In section Gallicanae, fourteen farm-raised and commercial samples were analyzed. The results of quantitative analysis and PCA indicated that the samples could be divided into three groups, because roses in section Gallicanae were complex hybrids of R. gallica, Rosa centifolia, and Rosa damascena, etc., and many horticultural roses are commercially available. Characteristics including contents of glycosides among the producing properties of these roses are influenced by the species used for breeding. 4. Concluding remarks In conclusion, the quantitative analysis of glycosides in the petals of Rosa species determined by UPLC provides a potent classification tool for sections Gallicanae, Cinnamomeae, Caninae and Synstylae.
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5.3. Extraction and isolation of flavonol glycosides Pulverized petals (348 g) collected in Hotan, Xiyumeigui farm, Xinjiang province in China (Ga01-1), were extracted five times with EtOH–H2O (8 l, 50:50, v/v) at room temperature for 24 h. After removal of the EtOH, the combined extracts were lyophilized. Then the resulting extract (150 g) was partitioned three times between MeOH:H2O:CHCl3 (1:1:2). The upper layer was evaporated to obtain crude material (135 g). The upper layer was suspended in H2O and was applied to a column of MCI gel CHP-20P (5 i.d. 47 cm). The column was eluted with a solvent system of H2O/MeOH/acetone to obtain fractions (Fraction 1–5). Fractions 2 (28 g) and 3 (12 g) were further purified by chromatography on Sephadex LH-20 and Wakosil 40 C18 columns to give compounds 1–13: compound 1 (120.0 mg), 2 (248.0 mg), 3 (423.0 mg), 4 (81.6 mg), 5 (4.63 mg), 6 (32.5 mg), 7 (9.95 mg), 8 (15.1 mg), 9 (264.2 mg), 10 (5.57 mg), 11 (20.9 mg), 12 (7.75 mg) and 13 (104.0 mg).
5. Experimental 5.1. General experimental procedures 1
H (500 MHz) and 13C NMR (125 MHz) spectra were measured with a JEOL a-500 spectrometer in CD3OD at 30 °C. Chemical shifts were determined using CD3OD (dH: 3.3 ppm, dC: 49.0 ppm) as the internal reference. APCI-TOF-MASS spectra were recorded on a JEOL JMS-T100LC spectrometer. Optical rotation was measured on a JASCO P-2200 polarimeter. MCI gel CHP-20 (Mitsubishi Chemical, Japan), Sephadex LH-20 (GE Healthcare, Sweden) and Wakosil 40 C18 (Wako Pure Chemical Industries, Ltd., Japan) were used for open column chromatography (CC). Ultra-performance liquid chromatography (UPLC) was performed using an Acquity UPLC system (Waters, USA) comprised of a binary solvent pump system, a sample manager, a column heater, a PDA detector, and an Acquity UPLC BEH C18 (2.1 i.d. 100 mm, 1.7 lm) column. All chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 5.2. Plant material Plants of R. gallica were not available from the wild, thus farmraised plants (Ga01-1–3) were collected in the Xinjiang province, China and commercially available hybrids of R. gallica were obtained from Yurigahara Lily Park, Sapporo (Ga03–07 and Ga10), and Chippubetsu Rose Garden, Chippubetsu, Hokkaido (Ga02, Ga09 and Ga11), Japan, respectively. Ga08 was the preserved sample of a hybrid of R. gallica collected at the Research Center for Medicinal Plant Resources National Institute of Biomedical Innovation, Nayoro, Hokkaido, Japan. Plants of R. rugosa growing in the wild were collected in Japan, Korea, and China, and wild plants of R. davurica were collected in Kamikawa, Hokkaido, Japan. Preservation plants belonging to section Cinnamomeae were collected at the Experimental Station for Medicinal Plant Studies Hokkaido University and Hokkaido (Ci04), Forestry Research Institute, Bibai, Hokkaido, (Ci02, Ci05 and Ci06-1), Japan. Two commercially available samples (Ci03 and Ci06-3) were obtained from Yurigahara Lily Park, Sapporo, Japan. Preservation samples of Rosa canica (Ca01) and Rosa multiflora (Sy01) were obtained from Hokkaido Forestry Research Institute, and two commercial samples (Ca02 and Sy02) were obtained from Yurigahara Lily Park, Sapporo, Japan. The species, collection reference, collection date, and facilities from which the samples are list below in Table 1. Fresh petals were separated and air-dried, and kept at room temperature in sealed package until used for experiments.
5.3.1. Quercetin 3-O-(200 -O-glucosyl)-b-D-galactoside (1) (Xiao et al., 2006) Brownish amorphous powder; [a]28 6.4° (c 3.0, MeOH); D Positive APCI-MS m/z 649.4 [M+Na]+. 5.3.2. Quercetin3-O-sophoroside (2) (Iwashina et al., 2005; Ross et al., 2005) Brownish amorphous powder; [a]28 D 23.1° (c 1.0, MeOH); Positive APCI-MS m/z 649.5 [M+Na]+. 5.3.3. Kaempferol3-O-sophoroside (3) (Schliemann et al., 2006; Ross et al., 2005) Yellow needles; [a]28 D 41.4° (c 1.0, MeOH); Positive APCI-MS m/z 633.5 [M+Na]+. 5.3.4. Quercetin3-O-b-D-galactoside (4, hyperoside) (Xiao et al., 2006; Zhu et al., 2013) Yellow needles; [a]28 D 9.1° (c 1.0, MeOH); Positive APCI-MS m/z 487.4 [M+Na]+. 5.3.5. Quercetin3-O-b-D-glucoside(5, isoquercitrin) Yellow needles; [a]28 D 5.0° (c 0.8, MeOH); Positive APCI-MS m/z 487.3 [M+Na]+. 5.3.6. Kaempferol3-O-b-D-galactoside (8, trifolin) Yellow needles; [a]28 D 15.7° (c 0.3, MeOH); Positive APCI-MS m/z 471.3 [M+Na]+. 5.3.7. Kaempferol3-O-b-D-glucoside (9, astragalin) (Xiao et al., 2006) Yellow needles; [a]28 D 11.4° (c 1.0, MeOH); Positive APCI-MS m/z 471.1 [M+Na]+. 5.3.8. Quercetin3-O-b-D-xylopyranoside (10, reynoutrin) (Zhu et al., 2013) Yellow amorphous powder; [a]28 D 17.8° (c 0.7, MeOH); Positive APCI-MS m/z 457.3 [M+Na]+. 5.3.9. Quercetin3-O-a-L-arabinofuranoside (11, avicularin) (Xiao et al., 2006; Park et al., 2012; Zhu et al., 2013) Brownish amorphous powder; [a]28 D 93.7° (c 1.0, MeOH); Positive APCI-MS m/z 457.3 [M+Na]+. 5.3.10. Kaempferol3-O-a-L-arabinofuranoside (12, juglanin) (Xiao et al., 2006; Zhu et al., 2013) Yellow needles; [a]28 D 121.1° (c 0.8, MeOH); Positive APCI-MS m/z 441.3 [M+Na]+.
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5.3.11. Kaempferol3-O-a-D-rhamnoside (13) (Nowak and Wolbis, 2002; Alaniya et al., 2012; Park et al., 2012) Yellow needles; [a]28 D 113.9° (c 0.4, MeOH); Positive APCI-MS m/z 455.2 [M+Na]+.
5.4. UPLC analysis Pulverized petals (1.00 g) of 31 samples used in this study, were individually extracted with EtOH:H2O (20 ml, 50:50, v/v) in an ultrasonic bath for 30 min. The supernatant was collected by centrifugation (1000g, 20 min), and was filtered through a 0.45 lm PTFE Millipore filter unit (Advantec, Japan), and then 1 ll of the filtrate was injected into the UPLC for analysis. The initial mobile phase (A) was CH3CN:H2O (2.5:97.5, v/v) containing 0.1% (v/v) HCO2H, and the final mobile phase (B) was CH3CN with 0.1% HCO2H. The gradient conditions were as follows: 0–4 min, 0% B; 26 min, 10% B; 45 min, 15% B; 51 min, 30% B; 54 min, 60% B; 55 min, 100% B. The flow rate was 0.2 ml/min. The detector wavelength range was set from 200 to 500 nm and the wavelength for quantitative determination was set at 350 nm.
5.5. Principal component analyses (PCA) PCA calculations were performed using IBM SPSSÒStatistics (Ver. 21.0). The contents of 15 flavonol glycosides and the percent ratio of four groups of flavonol glycosides classified by sugar moiety were used for original data.
Acknowledgments We thank Dr. Toshiro Shibata of the Research Center for Medicinal Plant Resources (Nayoro, Japan), National Institute of Biomedical Innovation, and Mr. Naotoshi Yoshida of the Hokkaido University, Medicinal Plant Garden, for kindly supplying the sample of Rosa spp. We also thank Mr. Guo Yong-Lai of the Pingying Rose Research Institute of China, and Prof. Mariya Sakim of the Xinjiang Medical University of China, for kindly supplying samples of Mei-gui.
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