Flavonoids from Sedum japonicum subsp. oryzifolium (Crassulaceae)

Abstract

Twenty-two flavonoids were isolated from the leaves and stems of Sedum japonicum subsp. oryzifolium (Crassulaceae). Of these compounds, five flavonoids were reported in nature for the first time, and identified as herbacetin 3-O-xyloside-8-O-glucoside, herbacetin 3-O-glucoside-8-O-(2′′′-acetylxyloside), gossypetin 3-O-glucoside-8-O-arabinoside, gossypetin 3-O-glucoside-8-O-(2′′′-acetylxyloside) and hibiscetin 3-O-glucoside-8-O-arabinoside via UV, HR-MS, LC-MS, acid hydrolysis and NMR. Other seventeen known flavonoids were identified as herbacetin 3-O-glucoside-8-O-arabinoside, herbacetin 3-O-glucoside-8-O-xyloside, gossypetin 3-O-glucoside-8-O-xyloside, quercetin, quercetin 3-O-glucoside, quercetin 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside, quercetin 3-O-rhamnoside-7-O-glucoside, kaempferol, kaempferol 3-O-glucoside, kaempferol 7-O-rhamnoside, kaempferol 3,7-di-O-rhamnoside, kaempferol 3-O-glucoside-7-O-rhamnoside, kaempferol 3-O-glucosyl-(1→2)-rhamnoside-7-O-rhamnoside, kaempferol 3-O-xylosyl-(1→2)-rhamnoside, kaempferol 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside, myricetin 3-O-glucoside and cyanidin 3-O-glucoside. Some flavonol 3,8-di-O-glycosides were found in Sedum japonicum subsp. oryzifolium as major flavonoids in this survey. They were presumed to be the diagnostic flavonoids in the species. Flavonoids were reported from S. japonicum for the first time.

1. Introduction

Sedum japonicum Siebold ex Miq. subsp. oryzifolium (Makino) H. Ohba (Crassulaceae) is distributed in Honshu, Shikoku, Kyushu and the Ryukyus in Japan and Korea and grows on rocks along the seacoast [1]. Various flavonoids, especially flavonols, have been reported from some Sedum species [2,3,4]. For example, thirty-four flavonoids including eight new flavonols, i.e., kaempferol 3-O-quinovosyl-(1→2)-rhamnoside-7-O-rhamnoside, quercetin 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside, kaempferol 3-O-[(6‴-E-p-coumaroylglucosyl)-(1→2)-glucoside]-7-O-rhamnoside, kaempferol 3-O-[(6‴-Z-p-coumaroylglucosyl)-(1→2)-glucoside]-7-O-rhamnoside, kaempferol 3-O-[glucosyl-(1→2)-(6″-acetylglucoside)]-7-O-rhamnoside and kaempferol 3-O-[(6‴-E-p-coumaroylglucosyl)-(1→2)-(6″-acetylglucoside)]-7-O-rhamnoside, have been isolated from Sedum bulbiferum Makino [5]. Thirty-one flavonoids including eight new flavonols, i.e., isorhamnetin 3-O-(6″-acetylglucoside)-7-O-glucoside, haplogenin 3-O-glucoside-7-O-rhamnoside, limocitrin 3-O-(6″-acetylglucoside)-7-O-glucoside, kaempferol 3-O-[(6‴-E-caffeoylglucosyl)-(1→2)-rhamnoside]-7-O-rhamnoside, quercetin 3-O-[(6‴-E-caffeoylglucosyl)-(1→2)-rhamnoside]-7-O-rhamnoside and isorhamnetin 3-O-rhamnoside-7-O-glucosyl-(1→2)-rhamnoside, have been reported from S. sarmentosum Bunge [6,7,8,9,10]. Thus, the presence of various new and rare flavonoids was presumed in Sedum species. In this survey, twenty-two flavonoids including five unreported compounds were isolated and identified from the leaves and stems of S. japonicum subsp. oryzifolium.

2. Results and Discussion

Twenty-two flavonoids were isolated from the leaves and stems of Sedum japonicum subsp. oryzifolium. Flavonoid 3 was obtained as a pale yellow powder, and demonstrated a molecular ion peak, m/z 595.1299 [M − H]⁻ calcd. for C₂₆H₂₇O₁₆ that appeared on HR-MS. Herbacetin, glucose and xylose were liberated via acid hydrolysis. Since a molecular ion peak, m/z 597 [M + H]⁺, and fragment ion peaks, m/z 435 [M-162 + H]⁺ and m/z 303 [M-162-132 + H]⁺, appeared on LC-MS, the attachment of each 1 mol of glucose and xylose to herbacetin was confirmed. In ¹H and ¹³C NMR, the proton and carbon signals were assigned via COSY, NOESY, HMQC and HMBC (

Table 1. ¹H (800 MHz) and ¹³C (200 MHz) NMR data (DMSO-d₆) of flavonoid glycosides from Sedum japonicum subsp. oryzifolium.

Positions	3		4		6		7		8
	δH	δC	δH	δC	δH	δC	δH	δC	δH	δC
	Herbacetin		Herbacetin		Gossypetin		Gossypetin		Hibiscetin
2		154.8		153.2		155.7		156.6		148.4
3		133.0		132.5		133.5		133.0		133.5
4		177.0		175.2		177.2		171.4		177.4
5		163.5		164.5		157.3		156.7		156.5
6	6.13 s	99.7	5.66 s	98.0	6.05 s	100.7	6.67 s	99.7	6.27 s	123.3
7		156.8		157.1		157.3		156.7		156.5
8		125.2		128.0		125.7		123.0		101.0
9		148.4		148.3		149.0		148.3		156.5
10		102.2		102.4		101.0		101.4		103.7
1′		121.1		121.6		122.0		121.5		120.1
2′	8.25 d (8.8)	131.1	8.21 d (8.8)	130.8	7.83 d (2.4)	117.6	7.79 d (1.6)	116.9	7.35 s	109.2
3′	6.85 d (8.8)	115.0	6.80 d (8.8)	114.7		145.2		144.5		145.3
4′		159.9		159.3		148.9		148.7		136.8
5′	6.85 d (8.8)	115.0	6.80 d (8.8)	114.7	6.81 d (8.8)	115.6	6.83 d (8.0)	115.2		145.3
6′	8.25 d (8.8)	131.1	8.21 d (8.8)	130.8	7.70 dd (2.4, 8.4)	122.1	7.59 brd (8.0)	121.3	7.35 s	109.2
	3-O-xylose		3-O-glucose		3-O-glucose		3-O-glucose		3-O-glucose
1	5.43 d (7.2)	101.3	5.33 d (8.0)	102.3	5.42 d (8.0)	102.0	5.41 d (8.0)	100.0	5.47 d (7.2)	106.2
2	3.34 t (8.8)	73.7	3.20 m	74.3	3.29 t (8.8)	74.6	3.22 t (8.4)	74.0	3.36 m	73.8
3	3.21 m	76.0	3.21 m	76.6	3.23 t (8.8)	77.1	3.18 m	76.6	3.23 m	76.6
4	3.41 m	69.3	3.12 m	69.8	3.14 t (5.6)	70.4	3.12 m	69.9	3.11 m	69.9
5a	3.14 t (5.6)	66.1	3.11 m	77.3	3.12 m	78.0	3.06 m	77.5	3.15 m	77.7
5b	3.79 dd (5.6, 11.6)
6a			3.38 m	60.9	3.36 dd (5.6, 12.0)	61.6	3.31 m	61.0	3.37 m	61.1
6b			3.59 m		3.60 brd (10.4)		3.59 brd (10.4)		3.62 m
	8-O-glucose		8-O-xylose		8-O-arabinose		8-O-xylose		8-O-arabinose
1	4.60 d (8.0)	106.6	5.56 brs	102.3	4.64 d (5.6)	106.3	5.00 d (4.0)	100.0	4.89 d (4.8)	103.8
2	3.23 m	74.2	4.89 m	76.8	3.70 m	71.1	5.12 t (4.8)	77.7	3.80 t (5.6)	70.4
3	3.21 m	76.5	3.20 m	76.5	3.50 m	72.6	3.21 m	76.6	3.61 m	71.4
4	3.11 m	69.9	3.55 m	71.4	3.69 m	67.2	3.44 m	70.1	3.45 dd (2.4, 11.2)	69.2
5a	3.11 m	77.5	3.64 m	68.3	3.46 brd (10.4)	65.7	3.70 m	66.1	3.44 m	64.0
5b			3.78 m		3.84 dd (4.0, 12.0)		3.85 m		3.85 dd (4.8, 11.6)
6a	3.36 m	60.9
6b	3.58 brd (11.2)
			2‴-acetic acid				2‴-acetic acid
COOH				170.2				169.5
CH3			2.06 s	21.1			2.02 s	20.9

, Figures S1–S5. The ¹H NMR spectrum of 3 demonstrated three aromatic proton signals, δ_H 8.25 (H-2′,6′), 6.85 (H-3′,5′) and 6.13 (H-6). Anomeric proton signals of glucose and xylose were observed at δ_H 5.43 (d, J = 7.2 Hz) and 4.60 (d, J = 8.0 Hz), respectively. The attachment of xylose to the 3-position of herbacetin was determined with HMBC correlation between the xylosyl anomeric proton signal at δ_H 5.43 and a C-3 carbon signal at δ_C 133.0. The glucosyl anomeric proton signal at δ_H 4.60 was correlated with the C-8 carbon signal at δ_C 125.2 using HMBC, showing the attachment of glucose to the 8-position of herbacetin. Since the coupling constants of anomeric proton signals of glucose and xylose were J = 8.0 and 7.2 Hz, they are β-forms [11]. Thus, 3 was identified as herbacetin 3-O-β-D-xylopyranoside-8-O-β-D-glucopyranoside (Figure 1). The compound was reported in nature for the first time [12,13].

Flavonoid 4 was obtained as a pale yellow powder, and herbacetin, glucose and xylose were produced via acid hydrolysis. However, since a molecular ion peak at m/z 637.1405 [M − H]⁻ calcd. for C₂₈H₂₉O₁₇ occurred with HR-MS, the attachment of 1 mol acetic acid to herbacetin was shown. In ¹H and ¹³C NMR, the proton and carbon signals were assigned via COSY, HMQC and HMBC (Table 1, Figure S2). The ¹H NMR spectrum of 4 demonstrated three aromatic proton signals at δ_H 8.21 (d, J = 8.8 Hz), 6.80 (d, J = 8.8 Hz) and 5.66 (s) corresponding to H-2′,6′, H-3′,5′ and H-6. Two anomeric proton signals were observed at δ_H 5.33 (d, J = 8.0 Hz) and 5.56 (brs), together with δ_H 2.06 (s) corresponding to acetyl CH₃. The attachment of glucose to the 3-position of herbacetin was observed via HMBC correlation between a glucosyl anomeric proton signal at δ_H 5.33 and a C-3 carbon signal at δ_C 132.5. On the other hand, the attachment of xylose to the 8-position of the aglycones was determined via HMBC correlation between the xylosyl anomeric proton signal at δ_H 5.56 and a C-8 carbon signal at δ_C 128.0. Moreover, it was demonstrated via HMBC correlation between an acetyl COOH carbon signal at δ_C 170.2 and a H-2 proton signal of xylose at δ_H 4.89 that the acetyl group is attached to the 2-position of xylose. Thus, 4 was identified as herbacetin 3-O-β-D-glucopyranoside-8-O-(2‴-acetylxyloside).

Flavonoid 6 demonstrated a molecular ion peak, m/z 611.1248 [M − H]⁻ calcd. for C₂₆H₂₇O₁₇ via HR-MS. In LC-MS, m/z 613 [M + H]⁺ and fragment ion peaks m/z 479 [M-132-H]⁻, m/z 451 [M-162 + H]⁺ and m/z 319 [M-162-132 + H]⁺ occurred, showing the attachment of each 1 mol of hexose and pentose to hexahydroxyflavone. Glucose and arabinose were liberated via acid hydrolysis, together with an aglycone. In ¹H and ¹³C NMR, the proton and carbon signals were assigned using COSY, NOESY, HMQC and HMBC (Table 1, Figure S3). The ¹H NMR spectrum of 6 demonstrated four aromatic proton signals at δ_H 7.83 (d, J = 2.4 Hz), 7.70 (dd, J = 2.4 and 8.4 Hz), 6.81 (d, J = 8.8 Hz) and 6.05 (s) corresponding to H-2′, H-6′, H-5′ and H-6, showing that the aglycone is 3,5,7,8,3′,4′-hexahydroxyflavone (gossypetin). Glucosyl and arabinosyl anomeric proton signals appeared at δ_H 5.42 (d, J = 8.0 Hz) and 4.64 (d, J = 5.6 Hz). In HMBC, the glucosyl anomeric proton signal was correlated with a C-3 carbon signal of gossypetin at δ_C 133.5. On the other hand, the arabinosyl anomeric proton signal was correlated with a C-8 carbon signal at δ_C 125.7. The coupling constants of anomeric proton signals of glucose and arabinose were J = 8.0 and 5.6 Hz, showing that they are β-D-pyranose and β-L-furanose, respectively [11]. Thus, 6 was identified as gossypetin 3-O-β-D-glucopyranoside-8-O-β-L-arabinofuranoside (Figure 1), which was found in nature for the first time.

Flavonoid 7 demonstrated a molecular ion peak, m/z 653.1354 [M − H]⁻ calcd. for C₂₈H₂₉O₁₈ using HR-MS. In LC-MS, molecular ion peaks, m/z 655 [M + H]⁺ and 653 [M − H]⁻, and fragment ion peaks, m/z 493 [M-162 + H]⁺ and m/z 319 [M-42-132-162 + H]⁺, occurred, showing the attachment of each 1 mol of hexose, pentose and acetic acid to hexahydroxyflavone. Glucose and xylose were liberated via acid hydrolysis, together with an aglycone. In ¹H and ¹³C NMR, the proton and carbon signals were assigned using COSY, NOESY, HMQC and HMBC (Table 1, Figure S4). The ¹H NMR spectrum of 7 demonstrated four aromatic proton signals at δ_H 7.79 (d, J = 1.6 Hz), 7.59 (brd, J = 8.0 Hz), 6.83 (d, J = 8.0 Hz) and 6.67 (s) corresponding to H-2′, H-6′, H-5′ and H-6, indicating that an aglycone is gossypetin. Anomeric proton signals of glucose and xylose were observed at δ_H 5.41 (d, J = 8.0 Hz) and 5.00 (d, J = 4.0 Hz), together with an acetyl CH₃ proton signal at δ_H 2.02. The attachment of glucose to the 3-position of gossypetin was confirmed via HMBC correlation between the glucosyl anomeric proton signal at δ_H 5.41 and a C-3 carbon signal at δ_C 133.0. On the other hand, the attachment of xylose to the 8-position of gossypetin was shown via HMBC correlation between a xylosyl anomeric proton signal at δ_H 5.00 and a C-8 carbon signal at δ_C 123.0. Moreover, it was demonstrated via HMBC correlation between an acetyl COOH carbon signal at δ_C 169.5 and a C-2 proton signal of xylose at δ_H 5.12 that acetyl group is attached to the C-2 of xylose. Since the coupling constants of anomeric proton signals of glucose and xylose were J = 8.0 and 4.0 Hz, they are β-D-pyranose and α-D-furanose, respectively [11]. Thus, 7 was identified as gossypetin 3-O-β-D-glucopyranoside-8-O-α-D-(2‴-acetylxylofuranoside), which was found in nature for the first time.

Flavonoid 8 demonstrated a molecular ion peak, m/z 627.1197 [M − H]⁻ calcd. for C₂₆H₂₇O₁₈ using HR-MS, showing the attachment of each 1 mol of hexose and pentose to heptahydroxyflavone. Glucose and arabinose were produced via acid hydrolysis, together with an aglycone. In ¹H and ¹³C NMR, the proton and carbon signals were assigned using COSY, NOESY, HMQC and HMBC (Table 1, Figure S5). Since two aromatic proton signals at δ_H 7.35 (2H, s) and 6.27 (1H, s) corresponding to H-2′,6′ and H-6 appeared on ¹H NMR, the aglycone was determined as 3,5,7,8,3′,4′,5′-heptahydroxyflavone (hibiscetin). Glucosyl and arabinosyl anomeric proton signals were found at δ_H 5.47 (d, J = 7.2 Hz) and 4.89 (d, J = 4.8 Hz). The attachment of glucose to the 3-position of the aglycone was confirmed via HMBC correlation between the glucosyl anomeric proton signal and a C-3 carbon signal at δ_C 133.5. The attachment of arabinose to the 8-position of the aglycone was confirmed via HMBC correlation between the arabinosyl anomeric proton signal and a C-8 carbon signal at δ_C 101.0. Since the coupling constants of the anomeric proton signals of glucose and arabinose were J = 7.2 and 4.8 Hz, they are β-D-pyranose and β-L-furanose, respectively [11]. Thus, 8 was identified as hibiscetin 3-O-β-D-glucopyranoside-8-O-β-L-arabinofuranoside (Figure 1). The compound was reported in nature for the first time.

Seventeen flavonoids (1, 2, 5, 9–22) were isolated from the leaves and stems of S. japonicum subsp. oryzifolium, together with five new compounds (3, 4, 6–8). Of these flavonoids, eight compounds were identified as herbacetin 3-O-glucoside-8-O-arabinoside (1), herbacetin 3-O-glucoside-8-O-xyloside (2), gossypetin 3-O-glucoside-8-O-xyloside (5), quercetin 3-O-rhamnoside-7-O-glucoside (12), quercetin 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside (11), kaempferol 3-O-glucosyl-(1→2)-rhamnoside-7-O-rhamnoside (18), kaempferol 3-O-xylosyl-(1→2)-rhamnoside (19) and kaempferol 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside (20) via UV spectral survey according to Mabry et al. [14], LC-MS, acid hydrolysis and NMR. Other flavonoids were characterized as quercetin (9), quercetin 3-O-glucoside (10), kaempferol (13), kaempferol 3-O-glucoside (14), kaempferol 7-O-rhamnoside (15), kaempferol 3,7-di-O-rhamnoside (16), kaempferol 3-O-glucoside-7-O-rhamnoside (17), myricetin 3-O-glucoside (21), and anthocyanin, cyanidin 3-O-glucoside (22) via UV spectra, LC-MS, acid hydrolysis, and HPLC and TLC comparisons with authentic samples. Kaempferol 7-O-rhamnoside (15) was characterized via UV, LC-MS and acid hydrolysis. These flavonoids were flavonols except for an anthocyanin, cyanidin 3-O-glucoside (22). Of these glycosides, eight (1–8) were 3,8-di-O-glycosides. Although flavonol 3,8-di-O-glycosides are comparatively rare flavonoids, they are sometimes reported from Crassulaceae species. Thus, herbacetin 3-O-glucoside-8-O-arabinoside, 3-O-arabinoside-8-O-xyloside and 3-O-rhamnoside-8-O-lyxoside, gossypetin 3-O-glucoside-8-O-xyoside and haplogenin 3-O-glucoside-8-O-xyloside were isolated from Phedimus aizoon (L.) ‘t Hart (= Sedum aizoon L.) [15]. Gossypetin 3-O-(3″-acetylglucoside)-8-O-glucuronide and herbacetin 3-O-(3″-acetylglucoside)-8-O-glucuronide and 3-O-glucoside-8-O-glucuronide were reported from Rhodiola quadrifida (Pall.) Fisch. & C.A. Mey [16]. Moreover, herbacetin 3-O-glucoside-8-O-xyloside was found in Rhodiola rosea L. [17,18,19,20]. In Sedum species, herbacetin 3-O-glucoside-8-O-xyloside was found in S. takesimense Nakai [21]. In this survey, herbacetin 3-O-glucoside-8-O-arabinoside (1), herbacetin 3-O-glucoside-8-O-xyloside (2), herbacetin 3-O-xyloside-8-O-glucoside (3), herbacetin 3-O-glucoside-8-O-(2‴-acetylxyloside) (4), gossypetin 3-O-glucoside-8-O-xyloside (5), gossypetin 3-O-glucoside-8-O-arabinoside (6), gossypetin 3-O-glucoside-8-O-(2‴-acetylxyloside) (7) and hibiscetin 3-O-glucoside-8-O-arabinoside (8) were found. Thus, flavonol 3,8-di-O-glycosides were presumed to be the diagnostic flavonoids in the Crassulaceae. We are now surveying other Crassulaceae species.

3. Materials and Methods

3.1. Plant Materials

Sedum japonicum Siebold ex Miq. subsp. oryzifolium (Makino) H. Ohba were collected in Kochi Pref., Shikoku, Japan in May–June 2021. Voucher specimens was deposited in the herbarium of the Kochi Prefectural Makino Botanical Garden, Kochi, Japan (MBK-0331366).

3.2. General

Analytical high performance liquid chromatography (HPLC) was performed with Shimadzu HPLC systems using Inertsil ODS-4 column (I.D. 6.0 × 150 mm, GL Science Inc., Tokyo, Japan) at a flow-rate of 1.0 mL/min. The detection wavelength was 350 nm. The eluent was MeCN/H₂O/H₃PO₄ [20:80:0.2 for glycosides (solv. I) and 40:60:0.2 for aglycones (solv. II)]. Liquid chromatograph-mass spectra (LC-MS) was performed with Shimadzu LC-MS systems using Inertsil ODS-4 column (I.D. 2.1 × 100 mm) at flow-rate of 0.2 mL/min, electrospray ionization (ESI⁺) 4.5 kV, ESI⁻ 3.5 kV, 250 °C. The eluent was MeCN/H₂O/HCOOH (17:78:5 for glycosides and 35:60:5 for aglycones). HR-MS (ESI⁻) was performed via JMS-T100LP mass spectrometer (JEOL Ltd., Tokyo, Japan). NMR spectroscopy was recorded on a JNM-ECZ800 spectrometer equipped with a 5-mm CH-UltraCOOL probe or on a JNM-ECA800 spectrometer equipped with a 5-mm HX-UltraCOOL probe (JEOL Ltd., Tokyo, Japan). All spectra were obtained in 0.2 mL of the deuterated solvent placed inside DMS-005J micro NMR tubes (SHIGEMI Co., Ltd., Tokyo, Japan) at 298 K. All samples were dissolved in a dimethyl sulfoxide-d₆ (DMSO-d₆: C₂D₆SO), 100.0 atom% D (Thermo Fischer Scientific, Waltham, MA, USA). The chemical shift was reported in parts per million (ppm) with coupling constants (J) in hertz relative to the solvent peaks; δ_H = 2.49 (residual C₂H₁D₅SO) and δ_C = 39.50 for C₂D₆SO, respectively. All NMR data reported in this article were obtained via ¹H NMR, ¹³C NMR, ¹H-¹H COSY, NOESY (mixing time: 450 ms), HMQC and HMBC experiments. Data analyses were performed using Delta NMR software (Ver. 6.0 or 6.1, JEOL Ltd.). NMR was also measured with a Bruker AV-600 spectrometer (Bruker Biospin AG, Switzerland) in DMSO-d₆. UV-visible absorption spectra were measured with a Shimadzu MPS-2000 multipurpose recording spectrophotometer. Acid hydrolysis was performed in 12% aq. HCl, 100 °C, 30 min. After shaking with diethyl ether, aglycones were migrated to the organic layer. On the other hand, sugars were left in the aqueous layer. Preparative HPLC was performed with Shimadzu HPLC systems using Inertsil ODS-4 column (I.D. 10 × 250 mm) at a flow-rate of 1.5 mL/min, detection wavelength of 350 nm, and eluent of MeCN/H₂O/HCOOH (20:75:5, 18:77:5 or 15:80:5). Preparative paper chromatography (prep. PC) was performed with solvent systems, BAW (n-BuOH/HOAc/H₂O = 4:1:5, upper phase) and then 15% HOAc. Analytical thin layer chromatography (TLC) was performed with solvent systems, BAW, BEW (n-BuOH/EtOH/H₂O = 4:1:2.2) and 15% HOAc.

3.3. Extraction and Isolation

Although four samples were collected in Kochi Prefecture, Japan, their flavonoid compositions were essentially the same with each other, which was recognized via analytical HPLC. Total fresh leaves and stems (ca. 1.0 kg) of S. japonicum subsp. oryzifolium were extracted with MeOH. After concentration, the extracts were applied to prep. PC using solvent systems, BAW and then 15% HOAc. Flavonoids 8–10, 13, 15, 16 and 19–22 were isolated, eluted with MeOH, and purified via Sephadex LH-20 column chromatography using solvent systems, 70% MeOH for flavonols and MeOH/H₂O/HCOOH (20:75:5) for anthocyanin. Other flavonoids, 1–7, 11, 12, 14, 17 and 18 were obtained as mixtures and separated with prep. HPLC. These flavonoids were obtained as pale yellow powders, i.e., 1 (8.6 mg), 2 (0.9 mg), 3 (8.4 mg), 4 (0.6 mg), 5 (6.4 mg), 6 (9.6 mg), 7 (0.8 mg), 8 (4.6 mg), 9 (trace), 10 (0.9 mg), 11 (3.7 mg), 12 (5.0 mg), 13 (trace), 14 (1.1 mg), 15 (0.6 mg), 16 (185.1 mg), 17 (1.0 mg), 18 (2.5 mg), 19 (1.6 mg), 20 (47.3 mg), 21 (0.6 mg) and 22 (trace).

3.4. Identification of Flavonoids

Flavonoids were identified via UV-vis spectral survey, HR-MS, LC-MS, acid hydrolysis, NMR and/or HPLC and TLC comparisons with authentic samples. NMR spectra and signal assignment for flavonoids are shown in Table 1 and Supplementary Materials Figures S1–S5. The origins of the authentic samples used in this survey were as follows: kaempferol from Dianthus caryophyllus flowers (as hydrolysate) [22], kaempferol 3-O-glucoside and quercetin 3-O-glucoside from Cyrtomium spp. fronds [23], kaempferol 3,7-di-O-rhamnoside from Hylotelephium sieboldii leaves and stems [24], kaempferol 3-O-glucoside-7-O-rhamnoside from Lathyrus japonicus leaves [25], quercetin and herbacetin from Extrasynthese (France), myricetin 3-O-glucoside from Corylopsis spp. leaves [26], and cyanidin 3-O-glucoside from Acer spp. leaves [27].

3.4.1. Herbacetin 3-O-glucoside-8-O-arabinoside (1)

TLC (Rf): 0.31 (BAW), 0.55 (BEW), 0.56 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark greenish yellow. HPLC (retention times, tR): 6.72 min (solv. I). UV: λmax (nm) MeOH 272, 357; +NaOMe 281, 326, 407 (inc.); +AlCl₃ 280, 310, 354, 407; +AlCl₃/HCl 280, 308, 347, 406; +NaOAc 280, 316, 398; +NaOAc/H₃BO₃ 274, 364. HR-MS (ESI) [M − H]⁻ calcd. for C₂₆H₂₇O₁₆: 595.1299, Found: 595.1291. LC-MS: m/z 597 [M + H]⁺, 595 [M − H]⁻, m/z 435 [M-162 + H]⁺ and m/z 303 [M-162-132 + H]⁺. ¹H NMR (800MHz, DMSO-d₆): δ 8.23 (2H, d, J = 8.8 Hz, H-2′,6′), 6.85 (2H, d, J = 8.8 Hz, H-3′,5′), 6.02 (1H, s, H-6), 5.41 (1H, d, J = 7.2 Hz, glucosyl H-1), 4.58 (1H, d, J = 6.4 Hz, arabinosyl H-1), 3.81 (1H, dd, J = 4.0 and 12.0 Hz, arabinosyl H-5b), 3.68 (1H, brd, J = 8.0 Hz, arabinosyl H-2), 3.67 (1H, brd, J = 6.4 Hz, arabinosyl H-4), 3.58 (1H, brd, J = 10.4 Hz, glucosyl H-6b), 3.48 (1H, brd, J = 10.4 Hz, arabinosyl H-3), 3.47 (1H, brd, J = 10.4 Hz, arabinosyl H-5a), 3.37 (1H, dd, J = 4.8 and 8.7 Hz, glucosyl H-6a), 3.22 (1H, m, glucosyl H-3), 3.21 (1H, m, glucosyl H-2), 3.11 (1H, m, glucosyl H-5), 3.10 (1H, m, glucosyl H-4). ¹³C NMR (200 MHz, DMSO-d₆): (herbacetin) δ 154.8 (C-2), 132.9 (C-3), 176.5 (C-4), 164.8 (C-5), 100.9 (C-6), 156.8 (C-7), 125.5 (C-8), 148.5 (C-9), 100.5 (C-10), 121.2 (C-1′), 131.0 (C-2′), 115.0 (C-3′), 159.9 (C-4′), 115.0 (C-5′), 131.0 (C-6′); (3-O-glucose) δ 101.6 (C-1), 74.3 (C-2), 76.6 (C-3), 69.9 (C-4), 77.5 (C-5), 60.9 (C-6); (8-O-arabinose) δ 106.3 (C-1), 70.7 (C-2), 72.3 (C-3), 67.0 (C-4), 65.5 (C-5).

3.4.2. Herbacetin 3-O-glucoside-8-O-xyloside (2)

TLC (Rf): 0.47 (BAW), 0.63 (BEW), 0.55 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ greenish yellow. HPLC (tR): 6.46 min (solv. I). UV: λmax (nm) MeOH 271, 351; +NaOMe 281, 325, 407 (inc.); +AlCl₃ 274, 308, 351, 405sh; +AlCl₃/HCl 275, 310, 350, 404sh; +NaOAc 280, 308, 384; +NaOAc/H₃BO₃ 273, 368. HR-MS (ESI) [M − H]⁻ calcd. for C₂₆H₂₇O₁₆: 595.1299, Found: 595.1278. LC-MS: m/z 597 [M + H]⁺, 595 [M − H]⁻, m/z 463 [M-132-H]⁻, m/z 435 [M-162 + H]⁺ and m/z 303 [M-162-132 + H]⁺. ¹H NMR (800MHz, DMSO-d₆): δ 8.24 (2H, d, J = 8.9 Hz, H-2′,6′), 6.82 (2H, d, J = 8.9 Hz, H-3′,5′), 5.75 (1H, s, H-6), 5.35 (1H, d, J = 7.6 Hz, glucosyl H-1), 4.34 (1H, d, J = 7.3 Hz, xylosyl H-1), 3.77 (1H, dd, J = 5.6 and 11.2 Hz, xylosyl H-5b), 3.58 (1H, brd, J = 11.2 Hz, glucosyl H-6b), 3.37 (1H, m, glucosyl H-6a), 3.34 (1H, m, xylosyl H-4), 3.19 (1H, m, glucosyl H-3), 3.18 (1H, m, xylosyl H-2), 3.18 (1H, m, glucosyl H-2), 3.18 (1H, m, xylosyl H-3), 3.10 (1H, m, glucosyl H-5), 3.10 (1H, m, xylosyl H-5a), 3.09 (1H, m, glucosyl H-4). ¹³C NMR (200 MHz, DMSO-d₆): (herbacetin) δ 153.4 (C-2), 132.6 (C-3), 175.7 (C-4), 164.0 (C-5), 101.7 (C-6), 157.1 (C-7), 127.2 (C-8), 148.3 (C-9), 101.7 (C-10), 121.6 (C-1′), 130.8 (C-2′), 114.8 (C-3′), 159.4 (C-4′), 114.8 (C-5′), 130.8 (C-6′); (3-O-glucose) δ 102.1 (C-1), 74.3 (C-2), 76.7 (C-3), 70.0 (C-4), 77.4 (C-5), 60.9 (C-6); (8-O-xylose) δ 108.4 (C-1), 73.8 (C-2), 76.6 (C-3), 69.2 (C-4), 66.3 (C-5).

3.4.3. Herbacetin 3-O-xyloside-8-O-glucoside (3)

TLC (Rf): 0.44 (BAW), 0.57 (BEW), 0.63 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark greenish yellow. HPLC (tR): 7.48 min (solv. I). UV: λmax (nm) MeOH 271, 355; +NaOMe 281, 327, 404 (inc.); +AlCl₃ 279, 309, 353, 407; +AlCl₃/HCl 279, 308, 348, 404; +NaOAc 280, 320, 400; +NaOAc/H₃BO₃ 274, 366. HR-MS (ESI) [M − H]⁻ calcd. for C₂₆H₂₇O₁₆: 595.1299, Found: 595.1290. LC-MS: m/z 597 [M + H]⁺, 595 [M − H]⁻, m/z 435 [M-162 + H]⁺ and m/z 303 [M-162-132 + H]⁺. ¹H and ¹³C NMR, see Table 1. Aglycone of 3 (herbacetin). HPLC (tR): 6.75 min (solv. II). UV: λmax (nm) MeOH 248, 275, 299, 370; +NaOMe decomp.; +AlCl₃ 260sh, 341, 378sh, 433; +AlCl₃/HCl 249, 268sh, 306, 365, 429; +NaOAc 274sh, 304, 372; +NaOAc/H₃BO₃ 310, 381.

3.4.4. Herbacetin 3-O-glucoside-8-O-(2‴-acetylxyloside) (4)

TLC (Rf): 0.59 (BAW), 0.71 (BEW), 0.56 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark greenish yellow. HPLC (tR): 15.49 min (solv. I). UV: λmax (nm) MeOH 272, 354; +NaOMe 282, 326, 408 (inc.); +AlCl₃ 274, 309, 354, 406sh; +AlCl₃/HCl 276, 308, 350, 405sh; +NaOAc 280, 309, 385; +NaOAc/H₃BO₃ 274, 319, 364. HR-MS (ESI) [M − H]⁻ calcd. for C₂₈H₂₉O₁₇: 637.1405, Found: 637.1380. LC-MS: m/z 639 [M + H]⁺, 637 [M − H]⁻, m/z 477 [M-162 + H]⁺ and m/z 303 [M-42-132-162 + H]⁺. ¹H and ¹³C NMR, see Table 1. Aglycone of 4 (herbacetin). HPLC (tR): 6.75 min (solv. II). UV: λmax (nm) MeOH 249, 276, 301, 374; +NaOMe decomp.; +AlCl₃ 267sh, 339, 374sh, 452; +AlCl₃/HCl 268, 306sh, 359, 436; +NaOAc 273sh, 323, 374; +NaOAc/H₃BO₃ 310, 377.

3.4.5. Gossypetin 3-O-glucoside-8-O-xyloside (5)

TLC (Rf): 0.28 (BAW), 0.49 (BEW), 0.48 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark greenish yellow. HPLC (tR): 5.34 min (solv. I). UV: λmax (nm) MeOH 260, 268sh, 364; +NaOMe 283, 418 (inc.); +AlCl₃ 278, 440; +AlCl₃/HCl 273, 304sh, 363, 410; +NaOAc 279, 328, 408; +NaOAc/H₃BO₃ 268, 388. LC-MS: m/z 613 [M + H]⁺, 611 [M − H]⁻, m/z 479 [M-132-H]⁻, m/z 451 [M-162 + H]⁺ and m/z 319 [M-162-132 + H]⁺. ¹H NMR (800 MHz, DMSO-d₆): δ 7.84 (1H, d, J = 2.4 Hz, H-2′), 7.75 (1H, dd, J = 2.4 and 8.8 Hz, H-6′), 6.80 (1H, d, J = 8.0 Hz, H-5′), 5.80 (1H, s, H-6), 5.38 (1H, d, J = 8.0 Hz, glucosyl H-1), 4.42 (1H, d, J = 8.0 Hz, xylosyl H-1), 3.83 (1H, dd, J = 4.8 and 11.2 Hz, xylosyl H-5b), 3.60 (1H, brd, J = 9.6 Hz, glucosyl H-6b), 3.38 (1H, m, xylosyl H-4), 3.37 (1H, t, J = 5.6 Hz, glucosyl H-6a), 3.28 (1H, t, J = 8.0 Hz, glucosyl H-3), 3.25 (1H, t, J = 8.0 Hz, xylosyl H-2), 3.23 (1H, t, J = 8.8 Hz, glucosyl H-2), 3.16 (1H, t, J = 8.8 Hz, xylosyl H-3), 3.12 (1H, m, glucosyl H-5), 3.11 (1H, m, xylosyl H-5a), 3.11 (1H, m, glucosyl H-4). ¹³C NMR (200 MHz, DMSO-d₆): (gossypetin) δ 154.4 (C-2), 132.8 (C-3), 176.1 (C-4), 157.0 (C-5), 101.0 (C-6), 157.0 (C-7), 126.5 (C-8), 148.4 (C-9), 99.8 (C-10), 121.6 (C-1′), 117.2 (C-2′), 144.6 (C-3′), 148.2 (C-4′), 114.9 (C-5′), 121.8 (C-6′); (3-O-glucose) δ 101.8 (C-1), 74.1 (C-2), 76.6 (C-3), 69.9 (C-4), 77.4 (C-5), 61.0 (C-6); (8-O-xylose) δ 107.9 (C-1), 73.8 (C-2), 76.5 (C-3), 69.2 (C-4), 66.2 (C-5).

3.4.6. Gossypetin 3-O-glucoside-8-O-arabinoside (6)

TLC (Rf): 0.21 (BAW), 0.44 (BEW), 0.46 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark greenish yellow. HPLC (tR): 5.74 min (solv. I). UV: λmax (nm) MeOH 262, 271, 365; +NaOMe 282, 420 (inc.); +AlCl₃ 274, 304sh, 370, 414sh; +AlCl₃/HCl 273, 305sh, 364, 407sh; +NaOAc 280, 327, 393; +NaOAc/H₃BO₃ 268, 390. HR-MS (ESI) [M − H]⁻ calcd. for C₂₆H₂₇O₁₇: 611.1248, Found: 611.1227. LC-MS: m/z 613 [M + H]⁺, 611 [M − H]⁻, m/z 479 [M-132-H]⁻, m/z 451 [M-162 + H]⁺ and m/z 319 [M-162-132 + H]⁺. ¹H and ¹³C NMR, see Table 1. Aglycone of 6 (gossypetin). HPLC (tR): 4.99 min (solv. II). UV: λmax (nm) MeOH 259, 280, 298, 341, 377; +NaOMe decomp.; +AlCl₃ 287, 336, 388, 466; +AlCl₃/HCl 273, 286sh, 304sh, 371, 443; +NaOAc 287, 375; +NaOAc/H₃BO₃ 310, 361.

3.4.7. Gossypetin 3-O-glucoside-8-O-(2‴-acetylxyloside) (7)

TLC (Rf): 0.51 (BAW), 0.63 (BEW), 0.64 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark greenish yellow. HPLC (tR): 10.13 min (solv. I). UV: λmax (nm) MeOH 261, 267sh, 360; +NaOMe 283, 325sh, 421 (inc.); +AlCl₃ 272, 369, 411; +AlCl₃/HCl 271, 304sh, 360, 408sh; +NaOAc 280, 325, 396; +NaOAc/H₃BO₃ 266, 384. HR-MS (ESI) [M − H]⁻ calcd. for C₂₈H₂₉O₁₈: 653.1354, Found: 653.1344. LC-MS: m/z 655 [M + H]⁺, 653 [M − H]⁻, m/z 493 [M-162-H]⁻, m/z 319 [M-42-132-162 + H]⁺. ¹H and ¹³C NMR, see Table 1. Aglycone of 7 (gossypetin). HPLC (tR): 4.91 min (solv. II). UV: λmax (nm) MeOH 261, 278, 304sh, 341, 381; +NaOMe decomp.; +AlCl₃ 288, 329sh, 383, 470; +AlCl₃/HCl 272, 309sh, 370, 445; +NaOAc 275sh, 376; +NaOAc/H₃BO₃ 305, 361.

3.4.8. Hibiscetin 3-O-glucoside-8-O-arabinoside (8)

TLC (Rf): 0.20 (BAW), 0.45 (BEW), 0.36 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark yellow. HPLC (tR): 5.01 min (solv. I). UV: λmax (nm) MeOH 268, 369; + NaOMe decomp.; +AlCl₃ 278, 443; +AlCl₃/HCl 277, 309, 368, 413; +NaOAc 279, 325, 420; +NaOAc/H₃BO₃ 268, 393. HR-MS (ESI) [M − H]⁻ calcd. for C₂₆H₂₇O₁₈: 627.1197, Found 627.1178. LC-MS: m/z 629 [M + H]⁺, 627 [M − H]⁻, m/z 495 [M-132-H]⁻, m/z 467 [M-162 + H]⁺ and m/z 335 [M-162-132 + H]⁺. ¹H and ¹³C NMR, see Table 1. Aglycone of 8 (hibiscetin). HPLC (tR): 3.95 min (solv. II). UV: λmax (nm) MeOH 242sh, 299, 360; +NaOMe decomp.; +AlCl₃ 259sh, 337, 406, 457sh; +AlCl₃/HCl 306, 373, 437sh; +NaOAc 308, 385; +NaOAc/H₃BO₃ 308, 387.

3.4.9. Quercetin (9)

TLC (Rf): 0.76 (BAW), 0.76 (BEW), 0.01 (15%HOAc); color UV (365 nm) yellow, UV/NH₃ yellow. HPLC (tR): 7.39 min (solv. II). UV: λmax (nm) MeOH 255, 273sh, 369; +NaOMe decomp.; +AlCl₃ 269, 449; +AlCl₃/HCl 263, 296sh, 357, 425; +NaOAc 274, 327, 400; +NaOAc/H₃BO₃ 258, 386. LC-MS: m/z 303 [M + H]⁺, 301 [M − H]⁻.

3.4.10. Quercetin 3-O-glucoside (Isoquercitrin, 10)

TLC (Rf): 0.67 (BAW), 0.73 (BEW), 0.23 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ yellow. HPLC (tR): 14.10 min (solv. I). UV: λmax (nm) MeOH 256, 266sh, 357; +NaOMe 275, 331, 409 (inc.); +AlCl₃ 275, 303sh, 434; +AlCl₃/HCl 269, 297sh, 361, 400; +NaOAc 273, 325, 400; +NaOAc/H₃BO₃ 261, 380. LC-MS: m/z 465 [M + H]⁺, 463 [M − H]⁻, m/z 303 [M-162 + H]⁺, 301 [M-162-H]⁻.

3.4.11. Quercetin 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside (11)

TLC (Rf): 0.51 (BAW), 0.68 (BEW), 0.84 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark yellow. HPLC (tR): 11.44 min (solv. I). UV: λmax (nm) MeOH 256, 265sh, 351; +NaOMe 273, 394 (inc.); +AlCl₃ 269, 404; +AlCl₃/HCl 269, 301sh, 356, 397sh; +NaOAc 257, 264sh, 358, 407sh; +NaOAc/H₃BO₃ 260, 368. LC-MS: m/z 725 [M − H]⁻, m/z 595 [M-132 + H]⁺, 593 [M-132-H]⁻, m/z 449 [M-132-146 + H]⁺, m/z 303 [M-132-146-146 + H]⁺. ¹H NMR (800 MHz, DMSO-d₆): δ 7.39 (1H, d, J = 2.4 Hz, H-2′), 7.30 (1H, dd, J = 2.4 and 8.4 Hz, H-6′), 6.89 (1H, d, J = 8.8 Hz, H-5′), 6.77 (1H, d, J = 1.6 Hz, H-8), 6.45 (1H, d, J = 2.4 Hz, H-6), 5.55 (1H, brs, 7-rhamnosyl H-1), 5.30 (1H, brs, 3-rhamnosyl H-1), 4.16 (1H, d, J = 8.0 Hz, xylosyl H-1), 4.07 (1H, brd, J = 3.2 Hz, 3-rhamnosyl H-2), 3.84 (1H, brs, 3-rhamnosyl H-3), 3.64 (1H, m, 7-rhamnosyl H-3), 3.61 (1H, m, 7-rhamnosyl H-5), 3.60 (1H, brd, J = 12.6 Hz, 7-rhamnosyl H-2), 3.43 (1H, m, xylosyl H-5b), 3.41 (1H, m, xylosyl H-4), 3.26 (1H, m, 3-rhamnosyl H-4), 3.19 (1H, m, 3-rhamnosyl H-5), 3.13 (1H, t, J = 9.6 Hz, 7-rhamnosyl H-4), 3.06 (1H, t, J = 8.8 Hz, xylosyl H-3), 2.95 (1H, t, J = 8.8 Hz, xylosyl H-2), 2.91 (1H, t, J = 11.2 Hz, xylosyl H-5a), 1.12 (3H, d, J = 6.2 Hz, 7-rhamnosyl CH₃), 0.92 (3H, d, J = 6.2 Hz, 3-rhamnosyl CH₃). ¹³C NMR (200 MHz, DMSO-d₆): (quercetin) δ 157.6 (C-2), 134.5 (C-3), 178.0 (C-4), 160.9 (C-5), 99.4 (C-6), 161.7 (C-7), 94.5 (C-8), 156.0 (C-9), 105.6 (C-10), 120.1 (C-1′), 115.5 (C-2′), 145.4 (C-3′), 149.1 (C-4′), 115.6 (C-5′), 121.0 (C-6′); (3-O-rhamnose) δ 101.0 (C-1), 80.6 (C-2), 69.8 (C-3), 71.6 (C-4), 69.3 (C-5), 17.4 (C-6); (7-O-rhamnose) δ 98.4 (C-1), 70.1 (C-2), 70.3 (C-3), 71.7 (C-4), 70.2 (C-5), 17.9 (C-6); (2″-O-xylose) δ 106.5 (C-1), 73.8 (C-2), 76.2 (C-3), 70.3 (C-4), 65.7 (C-5).

3.4.12. Quercetin 3-O-rhamnoside-7-O-glucoside (12)

TLC (Rf): 0.31 (BAW), 0.52 (BEW), 0.64 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark yellow. HPLC (tR): 4.69 min (solv. I). UV: λmax (nm) MeOH 256, 265sh, 350; +NaOMe 272, 395 (inc.); +AlCl₃ 274, 435; +AlCl₃/HCl 270, 297sh, 356, 397; +NaOAc 262, 395; +NaOAc/H₃BO₃ 260, 369. LC-MS: m/z 611 [M + H]⁺, m/z 609 [M − H]⁻, m/z 465 [M-146 + H]⁺, m/z 303 [M-146-162 + H]⁺. ¹H NMR (600 MHz, DMSO-d₆): δ 7.31 (1H, d, J = 2.2 Hz, H-2′), 7.26 (1H, dd, J = 2.2 and 8.3 Hz, H-6′), 6.87 (1H, d, J = 8.3 Hz, H-5′), 6.73 (1H, d, J = 2.1 Hz, H-8), 6.44 (1H, d, J = 2.1 Hz, H-6), 5.27 (1H, d, J = 1.2 Hz, 3-rhamnosyl H-1), 5.06 (1H, d, J = 7.6 Hz, 7-glucosyl H-1), 3.97 (1H, dd, J = 1.5 and 3.1 Hz, 7-glucosyl H-4), 3.69 (1H, brd, J = 10.4 Hz, 7-glucosyl H-6b), 3.50 (1H, dd, J = 3.2 and 9.2 Hz, 3-rhamnosyl H-5), 3.44 (1H, m, 7-glucosyl H-6a), 3.43 (1H, m, 7-glucosyl H-5), 3.29 (1H, m, 7-glucosyl H-3), 3.25 (1H, m, 7-glucosyl H-2), 3.18 (1H, m, 3-rhamnosyl H-3), 3.17 (1H, m, 3-rhamnosyl H-2), 3.13 (1H, m, 3-rhamnosyl H-4), 0.81 (3H, d, J = 6.1 Hz, 3-rhamnosyl CH₃). ¹³C NMR (150 MHz, DMSO-d₆): (quercetin) δ 157.9 (C-2), 134.5 (C-3), 177.9 (C-4), 160.9 (C-5), 99.3 (C-6), 162.9 (C-7), 94.5 (C-8), 156.1 (C-9), 105.7 (C-10), 120.5 (C-1′), 115.8 (C-2′), 145.3 (C-3′), 148.7 (C-4′), 115.5 (C-5′), 121.2 (C-6′); (3-O-rhamnose) δ 101.8 (C-1), 70.0 (C-2), 70.6 (C-3), 71.2 (C-4), 70.4 (C-5), 17.5 (C-6); (7-O-glucose) δ 99.9 (C-1), 73.1 (C-2), 76.4 (C-3), 69.6 (C-4), 77.2 (C-5), 60.6 (C-6).

3.4.13. Kaempferol (13)

TLC (Rf): 0.95 (BAW), 0.95 (BEW), 0.01 (15%HOAc); color UV (365 nm) yellow, UV/NH₃ yellow. HPLC (tR): 11.47 min (solv. II). UV: λmax (nm) MeOH 268, 367; +NaOMe decomp.; +AlCl₃ 270, 304sh, 350, 427; +AlCl₃/HCl 269, 301sh, 349, 427; +NaOAc 276, 313sh, 396; +NaOAc/H₃BO₃ 268, 370. LC-MS: m/z 287 [M + H]⁺, 285 [M − H]⁻.

3.4.14. Kaempferol 3-O-glucoside (Astragalin, 14)

TLC (Rf): 0.80 (BAW), 0.85 (BEW), 0.36 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark greenish yellow. HPLC (tR): 20.61 min (solv. I). UV: λmax (nm) MeOH 265, 349; +NaOMe 275, 323, 401 (inc.); +AlCl₃ 268, 303, 350, 402sh; +AlCl₃/HCl 269, 300, 348, 396sh; +NaOAc 272, 304, 373; +NaOAc/H₃BO₃ 264, 359. LC-MS: m/z 449 [M + H]⁺, 447 [M − H]⁻, m/z 287 [M-162 + H]⁺.

3.4.15. Kaempferol 7-O-rhamnoside (15)

TLC (Rf): 0.85 (BAW), 0.93 (BEW), 0.08 (15%HOAc); color UV (365 nm) yellow, UV/NH₃ yellow. HPLC (tR): 23.86 min (solv. I). UV: λmax (nm) MeOH 269, 370; +NaOMe decomp.; +AlCl₃ 271, 303sh, 361, 422; +AlCl₃/HCl 270, 301sh, 355, 423; +NaOAc 279, 316, 394; +NaOAc/H₃BO₃ 270, 375. LC-MS: m/z 433 [M + H]⁺, 431 [M − H]⁻, m/z 287 [M-146 + H]⁺.

3.4.16. Kaempferol 3,7-di-O-rhamnoside (16)

TLC (Rf): 0.79 (BAW), 0.81 (BEW), 0.71 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark yellow. HPLC (tR): 18.06 min (solv. I). UV: λmax (nm) MeOH 266, 342; +NaOMe 277, 383 (inc.); +AlCl₃ 276, 301, 347, 401; +AlCl₃/HCl 276, 299, 342, 399; +NaOAc 269, 391; +NaOAc/H₃BO₃ 268, 347. LC-MS: m/z 579 [M + H]⁺, 577 [M − H]⁻, m/z 433 [M-146 + H]⁺, 431 [M-146-H]⁻, m/z 287 [M-146-146 + H]⁺, 285 [M-146-146-H]⁻.

3.4.17. Kaempferol 3-O-glucoside-7-O-rhamnoside (17)

TLC (Rf): 0.52 (BAW), 0.70 (BEW), 0.64 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark greenish yellow. HPLC (tR): 10.67 min (solv. I). UV: λmax (nm) MeOH 265, 350; +NaOMe 275, 390 (inc.); +AlCl₃ 275, 299sh, 351, 398; +AlCl₃/HCl 275, 298sh, 348, 395; +NaOAc 268, 398; +NaOAc/H₃BO₃ 266, 356. LC-MS: m/z 595 [M + H]⁺, 593 [M − H]⁻, m/z 447 [M-146-H]⁻, 433 [M-162 + H]⁺, m/z 287 [M-162-146 + H]⁺.

3.4.18. Kaempferol 3-O-glucosyl-(1→2)-rhamnoside-7-O-rhamnoside (18)

TLC (Rf): 0.57 (BAW), 0.64 (BEW), 0.85 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark yellow. HPLC (tR): 13.61 min (solv. I). UV: λmax (nm) MeOH 265, 339; +NaOMe 273, 379 (inc.); +AlCl₃ 267, 299, 345, 400sh; +AlCl₃/HCl 268, 297sh, 340, 397sh; +NaOAc 265, 350; +NaOAc/H₃BO₃ 266, 352. LC-MS: m/z 739 [M − H]⁻, m/z 593 [M-146-H]⁻, m/z 433 [M-146-162 + H]⁺, m/z 287 [M-146-146-162 + H]⁺. ¹H NMR (600 MHz, DMSO-d₆): δ 7.80 (2H, d, J = 8.8 Hz, H-2′,6′), 6.92 (2H, d, J = 8.8 Hz, H-3′,5′), 6.78 (1H, d, J = 2.0 Hz, H-8), 6.44 (1H, d, J = 2.1 Hz, H-6), 5.55 (1H, brs, 3-rhamnosyl H-1), 5.54 (1H, brs, 7-rhamnosyl H-1), 4.23 (1H, d, J = 7.9 Hz, 2″-glucosyl H-1), 4.08 (1H, brd, J = 2.3 Hz, 2″-glucosyl H-4), 3.83 (1H, brs, 3-rhamnosyl H-3), 3.62 (1H, dd, J = 3.3 and 9.3 Hz, 3-rhamnosyl H-4), 3.54 (1H, brd, J = 8.5 Hz, 7-rhamnosyl H-3), 3.51 (1H, m, 2″-glucosyl H-6b), 3.43 (1H, m, 7-rhamnosyl H-5), 3.41 (1H, m, 2″-glucosyl H-6a), 3.40 (1H, m, 3-rhamnosyl H-2), 3.30 (1H, m, 3-rhamnosyl H-5), 3.29 (1H, m, 7-rhamnosyl H-4), 3.16 (1H, m, 2″-glucosyl H-3), 3.12 (1H, m, 2″-glucosyl H-5), 2.99 (1H, m, 7-rhamnosyl H-2), 2.98 (1H, m, 2″-glucosyl H-2), 1.11 (3H, d, J = 6.2 Hz, 7-rhamnosyl CH₃), 0.87 (3H, d, J = 6.2 Hz, 3-rhamnosyl CH₃). ¹³C NMR (150 MHz, DMSO-d₆): (kaempferol) δ 157.6 (C-2), 134.8 (C-3), 177.9 (C-4), 161.0 (C-5), 99.4 (C-6), 161.7 (C-7), 94.6 (C-8), 156.1 (C-9), 105.8 (C-10), 120.1 (C-1′), 130.7 (C-2′), 115.5 (C-3′), 160.4 (C-4′), 115.5 (C-5′), 130.7 (C-6′); (3-O-rhamnose) δ 101.0 (C-1), 81.2 (C-2), 70.1 (C-3), 71.6 (C-4), 69.3 (C-5), 17.9 (C-6); (7-O-rhamnose) δ 98.5 (C-1), 70.2 (C-2), 70.4 (C-3), 71.7 (C-4), 70.3 (C-5), 17.4 (C-6); (2″-O-glucose) δ 106.1 (C-1), 73.9 (C-2), 76.3 (C-3), 69.8 (C-4), 76.6 (C-5), 60.5 (C-6).

3.4.19. Kaempferol 3-O-xylosyl-(1→2)-rhamnoside (19)

TLC (Rf): 0.85 (BAW), 0.91 (BEW), 0.56 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark greenish yellow. HPLC (tR): 14.78 min (solv. I). UV: λmax (nm) MeOH 265, 337; +NaOMe 273, 322, 387 (inc.); +AlCl₃ 274, 303, 348, 390; +AlCl₃/HCl 275, 300, 340, 392; +NaOAc 274, 321, 381; +NaOAc/H₃BO₃ 266, 344. LC-MS: m/z 563 [M − H]⁻, m/z 433 [M-132 + H]⁺, m/z 287 [M-132-146 + H]⁺. ¹H NMR (800 MHz, DMSO-d₆): δ 7.76 (2H, d, J = 8.8 Hz, H-2′,6′), 6.92 (2H, d, J = 8.8 Hz, H-3′,5′), 6.34 (1H, brs, H-8), 6.15 (1H, brs, H-6), 5.38 (1H, brs, rhamnosyl H-1), 4.18 (1H, d, J = 7.2 Hz, xylosyl H-1), 4.01 (1H, brs, rhamnosyl H-2), 3.53 (1H, brd, J = 5.6 Hz, rhamnosyl H-3), 3.51 (1H, brd, J = 4.8 Hz, xylosyl H-5b), 3.42 (1H, dd, J = 4.8 and 8.8 Hz, xylosyl H-4), 3.20 (1H, m, rhamnosyl H-5), 3.11 (1H, t, J = 8.8 Hz, rhamnosyl H-4), 3.07 (1H, t, J = 8.8 Hz, xylosyl H-3), 2.96 (1H, t, J = 8.8 Hz, xylosyl H-2), 2.93 (1H, t, J = 11.2 Hz, xylosyl H-5a), 0.87 (3H, d, J = 6.4Hz, rhamnosyl CH₃). ¹³C NMR (200 MHz, DMSO-d₆): (kaempferol) δ 156.7 (C-2), 134.1 (C-3), 177.5 (C-4), 161.2 (C-5), 99.1 (C-6), 156.6 (C-7), 93.9 (C-8), 152.2 (C-9), 103.2 (C-10), 120.3 (C-1′), 130.4 (C-2′), 115.4 (C-3′), 161.1 (C-4′), 115.4 (C-5′), 130.4 (C-6′); (3-O-rhamnose) δ 100.7 (C-1), 80.5 (C-2), 70.3 (C-3), 71.6 (C-4), 69.3 (C-5), 17.4 (C-6); (2″-O-xylose) δ 106.4 (C-1), 73.7 (C-2), 76.2 (C-3), 70.3 (C-4), 65.8 (C-5).

3.4.20. Kaempferol 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside (20)

TLC (Rf): 0.44 (BAW), 0.67 (BEW), 0.85 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ dark yellow. HPLC (tR): 13.86 min (solv. I). UV: λmax (nm) MeOH 265, 340; +NaOMe 273, 378 (inc.); +AlCl₃ 275, 300, 344, 397; +AlCl₃/HCl 275, 297sh, 340, 395; +NaOAc 266, 385; +NaOAc/H₃BO₃ 265, 344. LC-MS: m/z 711 [M + H]⁺, 709 [M − H]⁻, m/z 579 [M-132 + H]⁺, 577 [M-132-H]⁻, m/z 433 [M-132-146 + H]⁺, m/z 287 [M-132-146-146 + H]⁺. ¹H NMR (800 MHz, DMSO-d₆): δ 7.81 (2H, d, J = 8.8 Hz, H-2′,6′), 6.94 (2H, d, J = 8.8 Hz, H-3′,5′), 6.79 (1H, d, J = 2.4 Hz, H-8), 6.46 (1H, d, J = 1.6 Hz, H-6), 5.55 (1H, brs, 7-rhamnosyl H-1), 5.38 (1H, brs, 3-rhamnosyl H-1), 4.18 (1H, d, J = 8.0 Hz, xylosyl H-1), 4.03 (1H, dd, J = 1.6 and 3.6 Hz, 3-rhamnosyl H-2), 3.81 (1H, brs, 3-rhamnosyl H-3), 3.63 (1H, m, 7-rhamnosyl H-3), 3.62 (1H, m, 7-rhamnosyl H-5), 3.56 (1H, m, 7-rhamnosyl H-2), 3.51 (1H, dd, J = 5.6 and 11.2 Hz, xylosyl H-5b), 3.43 (1H, dd, J = 5.6 and 9.2 Hz, xylosyl H-4), 3.30 (1H, t, J = 9.6 Hz, 3-rhamnosyl H-4), 3.22 (1H, m, 3-rhamnosyl H-5), 3.12 (1H, t, J = 8.8 Hz, 7-rhamnosyl H-4), 3.07 (1H, t, J = 9.6 Hz, xylosyl H-3), 2.96 (1H, t, J = 8.8 Hz, xylosyl H-2), 2.93 (1H, t, J = 11.2 Hz, xylosyl H-5a), 1.12 (3H, d, J = 6.4 Hz, 7-rhamnosyl CH₃), 0.89 (3H, d, J = 6.4 Hz, 3-rhamnosyl CH₃). ¹³C NMR (200 MHz, DMSO-d₆): (kaempferol) δ 157.6 (C-2), 134.6 (C-3), 178.0 (C-4), 161.0 (C-5), 99.5 (C-6), 161.7 (C-7), 94.7 (C-8), 156.1 (C-9), 105.7 (C-10), 120.1 (C-1′), 130.6 (C-2′), 115.5 (C-3′), 160.4 (C-4′), 115.5 (C-5′), 130.6 (C-6′); (3-O-rhamnose) δ 100.9 (C-1), 80.5 (C-2), 69.8 (C-3), 71.6 (C-4), 69.3 (C-5), 17.4 (C-6); (7-O-rhamnose) δ 98.4 (C-1), 70.1 (C-2), 70.4 (C-3), 71.7 (C-4), 70.2 (C-5), 17.9 (C-6); (2″-O-xylose) δ 106.4 (C-1), 73.7 (C-2), 76.3 (C-3), 70.3 (C-4), 65.8 (C-5).

3.4.21. Myricetin 3-O-glucoside (21)

TLC (Rf): 0.40 (BAW), 0.61 (BEW), 0.18 (15%HOAc); color UV (365 nm) dark purple, UV/NH₃ yellow. HPLC (tR): 8.77 min (solv. I). UV: λmax (nm) MeOH 257, 264sh, 360; +NaOMe decomp.; +AlCl₃ 272, 431; +AlCl₃/HCl 283, 309, 365sh, 402; +NaOAc 272, 325, 406; +NaOAc/H₃BO₃ 260, 300, 382. LC-MS: m/z 481 [M + H]⁺, 479 [M − H]⁻, m/z 319 [M-162 + H]⁺.

3.4.22. Cyanidin 3-O-glucoside (Chrysanthemin, 22)

HPLC (tR): 4.19 min (solv. I). UV: λmax (nm) 0.01%HCl-MeOH 277, 332, 528; +AlCl₃ 275, 537; E₄₄₀/E_max = 26.5%. LC-MS: m/z 449 [M]⁺, m/z 287 [M-162]⁺.

4. Conclusions

Twenty-two flavonoids were isolated from the leaves and stems of Sedum japonicum subsp. oryzifolium (Crassulaceae). Of these compounds, five flavonoids were reported in nature for the first time, and identified as herbacetin 3-O-xyloside-8-O-glucoside, herbacetin 3-O-glucoside-8-O-(2‴-acetylxyloside), gossypetin 3-O-glucoside-8-O-arabinoside, gossypetin 3-O-glucoside-8-O-(2‴-acetylxyloside) and hibiscetin 3-O-glucoside-8-O-arabinoside via UV, HR-MS, LC-MS, acid hydrolysis and NMR. Some flavonol 3,8-di-O-glycosides were found in Sedum japonicum subsp. oryzifolium as major flavonoids in this survey, and they were presumed to be the diagnostic flavonoids in the species. Flavonoids were reported from S. japonicum for the first time.