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New triterpenoid saponins from the flowers of Pueraria thomsonii a
b
a
a
a
Jing Lu , Jia-Hong Sun , Yao Tan , Yoshihiro Kano & Dan Yuan a
Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang, 110016, China b
Department of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 110016, China Published online: 29 Oct 2013.
To cite this article: Jing Lu, Jia-Hong Sun, Yao Tan, Yoshihiro Kano & Dan Yuan (2013) New triterpenoid saponins from the flowers of Pueraria thomsonii, Journal of Asian Natural Products Research, 15:10, 1065-1072, DOI: 10.1080/10286020.2013.802690 To link to this article: http://dx.doi.org/10.1080/10286020.2013.802690
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Journal of Asian Natural Products Research, 2013 Vol. 15, No. 10, 1065–1072, http://dx.doi.org/10.1080/10286020.2013.802690
New triterpenoid saponins from the flowers of Pueraria thomsonii Jing Lua, Jia-Hong Sunb, Yao Tana, Yoshihiro Kanoa and Dan Yuana* a
Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016, China; bDepartment of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
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(Received 4 March 2013; final version received 1 May 2013) Two new oleanane-type triterpenoid saponins, kakkasaponin II (1) and kakkasaponin III (2), were isolated from the methanol extract of the flowers of Pueraria thomsonii (Leguminosae), together with seven known oleanane-type triterpenoid saponins, phaseoside IV (3), sophoradiol monoglucuronide (4), kakkasaponin I (5), kaikasaponin III (6), soyasaponin I (7), soyasaponin III (8), and soyasaponin IV (9). The structures of 1 and 2 were elucidated by spectroscopic methods including IR, ESI-TOF-MS, and 1D and 2D NMR experiments. Keywords: Pueraria thomsonii; Leguminosae; triterpenoid saponins
1.
Introduction
Puerariae Flos, a folk herb, is used to treat the complication of diabetes and alcohol intoxication in China, Korea, and Japan [1– 3], and similarly, the roots of the plant are a well-known herbal medicine mentioned in Chinese and Japanese Pharmacopoeias [4,5]. Puerariae Flos is botanically from the flowers of Pueraria lobata (Willd.) Ohwi and Pueraria thomsonii Benth. (Leguminosae). Previous phytochemical studies on flowers of both Pueraria sp. reported the isolation of many saponins and isoflavones [6–10]. The isoflavones, such as kakkalide, tectoridin, and tectorigenin, possess hepatoprotective, hypoglycemic, hypolipidemic, antioxidant and antiinflammatory activities [11–15], meanwhile the saponins, such as kakkasaponin I, kaikasaponin III, and soyasaponin I, possess hepatoprotective, hypoglycemic, hypolipidemic, antioxidant, and anticomplementary activities [7,14,16,17]. The HPLC profile reported by Kinjo et al. and Niiho et al. [7,18] showed that the major saponins, kakkasaponin I, kaikasaponin III,
and soyasaponin I, are found in both P. lobata and P. thomsonii flowers. However, the minor saponins from both flowers have not yet been studied. In terms of resource utilization, further investigation on the saponins of P. thomsonii flowers is very important because of its richer botanical nature than that of P. lobata flowers [19]. This study describes the isolation and structural elucidation of two new oleananetype triterpenoid saponins (1 and 2) and seven known saponins (3 – 9) from P. thomsonii flowers. 2. Results and discussion The MeOH extract of P. thomsonii flowers was prepared by the reflux method. The EtOAc and n-BuOH fractions were obtained by partitioning the MeOH extract. Highperformance liquid chromotography diode array detection (HPLC-DAD) analysis (data not shown) indicated that the EtOAc fraction mainly contained tectoridin derivatives, while the n-BuOH fraction mainly contained saponins as well as minor tectoridin derivatives. Further isolation of
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
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the n-BuOH fraction led to two new compounds, kakkasaponin II (1) and kakkasaponin III (2), together with seven known triterpenoid saponins, phaseoside IV (3), sophoradiol monoglucuronide (4), kakkasaponin I (5), kaikasaponin III (6), soyasaponin I (7), soyasaponin III (8), and soyasaponin IV (9) (Figure 1). The structures of compounds 1 and 2 were elucidated by IR, ESI-TOF-MS, and 1D and 2D NMR techniques. Compounds 3–9 were identified by comparing their NMR and MS data with reported values [6,17,20–23], among which, compounds 3 and 9, or compounds 4 and 8 were for the first time isolated from Pueraria sp. or from P. thomsonii flowers, respectively. The inhibitory activities of the isolates on AR (aldose reductase) from bovine lens have been assayed. Nevertheless, all of them showed little activity (data not shown), and the bioactivities of triterpenoid saponins from P. thomsonii flowers need to be investigated in subsequent research. Compound 1 was isolated as an amorphous powder and showed positive results to the Molish and Lieberman–Burchard tests. The molecular formula was determined to be C42H66O13 from a pseudomolecular ion peak at m/z 777.4423 [M–H] – in the negative ESITOF-MS. Two important fragment ion peaks at m/z 597.4 [M–C6H10O5 –H2O–H] – and 439.4 [M–C12H18O11 –H] – suggested the
Figure 1. Structures of triterpenoid saponins 1 – 9.
presence of a glucuronosyl and a hexosyl group. Further loss of 28 amu [–CO] from it produced a fragment ion peak at m/z 411.4, suggesting the presence of a carbonyl group. The IR spectrum showed absorption bands due to a hydroxyl (3448 cm21), a carboxylic carbonyl (1700 cm21), and an alkenyl (1636 cm21). The 1H and 13C NMR spectra (Table 1) showed typical signals for an oleanene-type sapogenol, including eight methyl groups (dH 0.83, 0.85, 0.89, 0.93, 1.13, 1.16, 1.27, and 1.33, 3H each, s), an olefinic proton (dH 5.23, t, J ¼ 3.78 Hz), two olefinic carbons (dC 123.1 and 141.9), a carbonyl group (dC 215.7), and an oxygenated carbon (dC 89.1). The location of the double bond at C12 –C13 and the carbonyl group at C22 was confirmed by the HMBC correlations between H-27 and C-13 and between H-28 and C-22, respectively. The resonances for the carbons and protons located on the aglycone framework had a close resemblance to those of the known abrisapogenol F, and they were assigned according to the 1H and 13C NMR data as well as the 2D NMR data reported in the literature for abrisapogenol F [21]. The carbohydrate chain of 1 consisted of two monosaccharide residues, as deduced from the signals of two anomeric carbons (dC 105.4 and 107.3) in the 13C NMR spectrum. Both anomeric carbons were correlated with the corresponding signals of anomeric
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Table 1. NMR (600 MHz) data of compound 1 in C5D5N (d in ppm, J in Hz).
dC
dH
1
38.7 (t)
2
26.7 (t)
3 4 5 6 7
89.1 (d) 39.6 (s) 55.7 (d) 18.4 (t) 32.9 (t)
1.37 – 1.39 (m, Hax) 0.79 – 0.83 (m, Heq) 1.88 – 1.90 (m, Hax) 2.21 – 2.25 (m, Heq) 3.31 (dd, J ¼ 11.5, 4.6, 1H)
8 9 10 11 12 13 14 15 16
39.9 (s) 47.7 (d) 36.8 (s) 23.8 (t) 123.1 (d) 141.9 (s) 42.0 (s) 25.4 (t) 27.4 (t)
17 18 19
47.9 (s) 47.9 (d) 46.6 (t)
20 21
34.2 (s) 50.9 (t)
HMBCa
COSYb
H-3 C-10
Hax-2
0.72 (d, J ¼ 11.4, 1H) 1.47 – 1.49 (m, Heq), 1.28c 1.39 – 1.42 (m, Hax) 1.23 – 1.26 (m, Heq)
C-25
Hax-6 H-5
1.57 (t, J ¼ 9.6, 1H)
C-10
1.77 – 1.83 (m, 2H) 5.23 (br s) 1.63 – 1.66 (m, Hax), 0.97c 2.08 – 2.11 (m, Hax) 1.14c 2.35 (dd, J ¼ 14.8, 3.7, 1H) 2.14 – 2.17(m, Hax), 1.29c 2.11 – 2.15 (m, Hax) 2.58 (d, J ¼ 13.8, Heq)
C-18, C-20, C-30 C-13
Hax, Heq-19 H-18 H-18
C-19, C-20, C-30 C-20, C-22, C-29
22 23 24 25 26 27 28 29 30 Glu 10 20 30 40 50 60 Gal 100 200 300 400 500 600
215.7 (s) 28.2 (q) 16.8 (q) 15.7 (q) 16.9 (q) 25.6 (q) 21.0 (q) 31.9 (q) 25.3 (q) 105.4 (d) 84.0 (d) 77.5 (d) 74.8 (d) 77.7 (d) 172.9 (s) 107.3 (d) 73.2 (d) 75.0 (d) 69.5 (d) 77.0 (d) 61.3 (t)
1.33 (s, 3H) 1.13 (s, 3H) 0.85 (s, 3H) 0.89 (s, 3H) 1.27 (s, 3H) 1.16 (s, 3H) 0.93 (s, 3H) 0.83 (s, 3H) 5.04 (d, J ¼ 7.8, 1H) 4.31 – 3.35 (m, 1H) 4.62c 4.63 – 4.66 (m, 1H) 4.38 – 4.40 (m, 1H) n.d. 5.25 (d, J ¼ 7.8, 1H) 4.60c 4.20 – 4.22 (m, 1H) 4.73 – 4.74 (m, 1H) 4.08 – 4.10 (m, 1H) 4.42 – 4.44 (m, 1H) 4.67c
C-3, C-4, C-5, C-24 C-3, C-4, C-5, C-23 C-5, C-10 C-7, C-8, C-9, C-14 C-8, C-13, C-14 C-17, C-18, C-22 C-19, C-20, C-21 C-19, C-20, C-21 C-3 C-10 , C-30 C-40
C-20 C-300 C-600
H-20 H-10
H-200 H-100 , H-300 H-200 , H-400 H-300 H-600 H-500
Notes: All chemical shift assignments were carried out on the basis of HSQC and HMBC NMR techniques. n.d., not determined. a HMBC correlations, optimized for 7 Hz, are from proton(s) stated to the indicated carbon. b1 H– 1H COSY correlations. c Overlapped with other signals, chemical shift obtained from 2D correlation.
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protons (dH 5.04, d, J ¼ 7.8 Hz and 5.25, d, J ¼ 7.8 Hz) by the heteronuclear singlequantum coherence (HSQC) spectrum. The signals of a glucuronide and a galactose residue were also identified, while the coupling constants of the anomeric protons indicated b-configuration of both glycosidic bonds [20]. All sugars of glycosides from the leaves of P. lobata belong to the D -series, as established earlier [19]. The position of interglycosidic linkage was confirmed on the basis of the HMBC correlation between H-1 of the galactose and C-2 of the glucuronide residue (Figure 2). The linkage of the carbohydrate moiety to aglycone was determined by HMBC correlation between H-1 of the glucuronic acid residue and C-3 of the aglycone, as well as by the downfield shift of C-3 (þ10.2 ppm) and the upfield shift of H-3a (–1.21 ppm) relative to the corresponding signals of abrisapogenol F [21]. Acid hydrolysis of 1 with 3N HCl yielded glucuronic acid, galactose, and aglycone abrisapogenol F, which were identified by co-thin layer chromatography (TLC) with authentic samples. Therefore, the structure of 1 was determined to be abrisapogenol F 3-O-b-D -galactopyranosyl(1 ! 2)-b-D -glucuropyranoside, named as kakkasaponin II.
Compound 2 was isolated as a white amorphous powder and showed positive results to the Molish and Lieberman– Burchard tests. The molecular formula was determined to be C47H74O16 from a pseudomolecular ion peak at m/z 893.4897 [M–H] – in negative ESI-TOF-MS. Four important fragment ion peaks at m/z 747.4 [M–C6H10O4 –H] – , 597.4 [M–C11H18O8 – H2O – H] – , 439.4 [M – C17H26O14 – H] – , and 411.4 [M – C17H26O14 – CO – H] – suggested the presence of a hexosyl, a pentosyl, a glucuronosyl, and a carbonyl group. The 1H and 13C NMR data (Table 2) together with the aglycone ion peak at m/z 439.4 indicated that the aglycone part of 2 was the same as that of 1. The carbohydrate chain consisted of three monosaccharide residues, as deduced from the signals of three anomeric carbons (dC 102.4, 102.7, and 105.2). These signals were correlated with the corresponding signals of anomeric protons (dH 6.39, br s; 5.76, d, J ¼ 7.3 Hz and 4.94, d, J ¼ 7.8 Hz) by the HSQC spectrum. The chemical shifts and coupling constants of the anomeric protons indicated a b-glucuronosyl, a b-xylopyranosyl group, and the remaining anomeric carbon at dC 102.4 was determined as a for rhamnose residue, whose NMR data were similar to
Figure 2. Key HMBCs of the sugar portion of compounds 1 and 2.
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Table 2. NMR (600 MHz) data of compound 2 in C5D5N (d in ppm, J in Hz). dC
dH
1 2
38.8 (t) 26.5 (t)
3 4 5 6
89.9 39.7 55.7 18.4
(d) (s) (d) (t)
0.85–0.88 (m, Heq), 1.39c 1.81–1.85 (m, Hax) 2.14–2.17 (m, Heq) 3.23 (dd, J ¼ 10.6, 3.6, 1H)
7 8 9 10 11 12 13 14 15
32.9 39.9 47.8 36.8 23.8 123.8 141.9 42.0 25.4
(t) (s) (d) (s) (t) (d) (s) (s) (t)
16
27.4 (t)
17 18 19
47.9 (s) 47.9 (d) 46.6 (t)
20 21
34.2 (s) 50.9 (t)
22 23 24 25 26 27 28 29 30 Glu 10 20 30 40 50 60 Xyl 100 200 300 400 500
215.7 (s) 28.3 (q) 16.7 (q) 15.7 (q) 16.9 (q) 25.5 (q) 21.0 (q) 31.9 (q) 25.3 (q) 105.2 (d) 78.7 (d) 77.0 (d) 74.0 (d) 77.7 (d) n.d. 102.7 (d) 79.3 (d) 78.1 (d) 71.0 (d) 66.8 (t)
Rha 1000 2000 3000 4000 5000 6000
102.4 72.4 72.6 74.3 69.6 19.0
(d) (d) (d) (d) (d) (q)
0.76 (d, J ¼ 12.0, 1H) 1.52–1.54 (m, Heq) 1.31c 1.23–1.25 (m, Heq), n.d.
HMBCa
COSYb H-3 H-3 Hax, Heq-2
C-25
Hax-6 H- 5
1.62 (t, J ¼ 8.4, 1H) 1.85–1.90 (m, 2H) 5.24c 1.66–1.69 (m, Hax) 0.97–0.99 (m, Heq) 2.06–2.08 (m, Hax) 1.11c
Hax-16
2.36 (dd, J ¼ 12.0, 3.8, 1H) 2.12–2.14 (m, Hax) 1.29c
C-13
Hax, Heq-19 H-18 H-18
2.08–2.12 (m, Hax) 2.58 (d, J ¼ 13.8, Heq)
C-19, C-20, C-30 C-22, C-29
1.33 (s, 3H) 1.08 (s, 3H) 0.84 (s, 3H) 0.90 (s, 3H) 1.28 (s, 3H) 1.16 (s, 3H) 0.94 (s, 3H) 0.83 (s, 3H) 4.94 (d, J ¼ 7.8, 1H) 4.56c n.d. 4.36–4.39 (m, 1H) 4.28–4.31 (m, 1H) n.d. 5.76 (d, J ¼ 7.3, 1H) 4.15–4.17 (m, 1H) 4.36–4.39 (m, 1H) 4.17–4.21 (m, 1H) 3.53–3.56 (m, 1H) 4.29–4.33 (m, 1H) 6.39 (br s, 1H) 4.77c 4.70c 4.33–4.36 (m, 1H) 5.07c 1.77 (d, J ¼ 6.0, 3H)
C-3, C-4, C-5, C-24 C-3, C-4, C-5, C-23 C-5, C-10 C-7, C-8, C-9, C-14 C-8, C-13, C-14 C-17, C-18, C-22 C-19, C-20, C-21 C-19, C-20, C-21 C-3
Hax-15
H-40 H-20 C-20
C-400 C-200 , C-2000 , C-5000
C-4000 , C-5000
H-500 H-500 H-400 H-100 H-2000 H-1000 H-4000 H-3000 , H-5000 H-4000 , H-6000 H-5000
Note: All chemical shift assignments were carried out on the basis of HSQC and HMBC NMR techniques. n.d., not determined. a HMBC correlations, optimized for 7 Hz, are from proton(s) stated to the indicated carbon. b H – 1H COSY correlations. c Overlapped with other signals, chemical shift obtained from 2D correlation.
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those observed in baptisiasaponin I isolated from the roots of Baptisia australis (Leguminosae) earlier [24]. The interglycosidic linkages were confirmed by the HMBC correlations between H-1 of the xlyose and C-2 of the glucuronide residue and between H-1 of the rhamnose and C-2 of the xylose residue (Figure 2). The linkage of the carbohydrate moiety to aglycone was determined by HMBC correlation between H-1 of the glucuronic acid residue and C-3 of the aglycone, as well as by the downfield shift of C-3 (þ11.0 ppm) and the upfield shift of H-3a (–1.27 ppm) relative to the corresponding signals of abrisapogenol F [21]. Acid hydrolysis of 2 with 3N HCl yielded glucuronic acid, xlyose, rhamnose residues (identified by co-TLC with authentic samples) as well as the aglycone abrisapogenol F. Therefore, the structure of 2 was determined to be abrisapogenol F 3O-a-L -rhamnopyrannosyl-(1 ! 2)-b-D xylopyranosyl-(1 ! 2)-b-D -glucuropyranoside, named as kakkasaponin III. 3. 3.1
Experimental General experimental procedures
Optical rotations were recorded on a Perkin-Elmer 241MC automatic polarimeter (Perkin-Elmer, Waltham, MA, USA). IR spectra were obtained on a Bruker IFS-55 infrared spectrometer (Bruker, Faellanden, Switzerland). 1D and 2D NMR spectra were recorded separately on a Bruker ARX-600 or ARX-300 spectrometer (Bruker). ESI-TOF-MS were acquired on a Micro TOF Bruker Daltonics mass spectrometer with a resolution of 25,000 (10% Valley, Bruker, Karlsruhe, Germany) and ESI-MS were acquired on a Shimadzu QP8000a mass spectrometer (Shimadzu Corporation, Shimadzu, Japan). Preparative HPLC was carried out using a Shimadzu’s LC-6A or Shimadzu’s LC-8A solvent delivery pump and Shimadzu’s SPD-10AVP detector (Shimadzu Corporation). Column chromatography (CC) was carried out on
silica gel (200 – 300 mesh, Qingdao Haiyang Chemical Group Co., Qingdao, China), Sephadex LH-20 (GE Healthcare, Uppsala, Sweden), octadecylsilyl silica (ODS) (YMC Co., Ltd, Kyoto, Japan), MDS-5 reversed-phase packing (200 – 300 mesh, Beijing Medicine Technology Center, Beijing, China), and Waters C18 column (7.8 mm £ 250 mm, 6 mm, Waters Co., Ltd, Milford, MA, USA). TLC was carried out on Merck silica gel 60F254 plates (E. Merck, Darmstadt, Germany). Other chemical reagents were of analysis or HPLC grades. Double distilled water was used in this study. The authentic sugars were purchased from SigmaAldrich (St Louis, MO, USA). 3.2 Plant material The flowers of P. thomsonii were collected in October 2006 in Hengyang, Hunan Province, China. Species identification was confirmed by Prof. Wei-Ning Wang, Liaoning Institute for Food and Drug Control, China. A voucher specimen (A0270610F) has been deposited in the Department of Traditional Chinese Medicines, Shenyang Pharmaceutical University. 3.3 Extraction and isolation The air-dried flowers (7.0 kg) of P. thomsonii were refluxed with MeOH (2 £ 42 liters; each 3 h) and evaporated in vacuo. The MeOH extract (1030 g) was partitioned between hexane (3 £ 3 liters) and H2O (3 £ 3 liters), and the H2O phase was extracted with EtOAc (3 £ 3 liters) to give an EtOAc fraction. The H2O phase was further extracted with n-BuOH (3 £ 3 liters) to give an n-BuOH fraction. The dried n-BuOH fraction was subjected to CC (silica gel, 1.5 kg), gradient of CHCl3 – MeOH – H2 O, 70:25:3 ! 60:40:10) to give Frs 1 – 8. These fractions were further subjected to repeated CC (MDS-5, gradient of MeOH –H2O, 50:50 ! 90:10; ODS, gradient of MeOH – H2O, 60:40 ! 80:20).
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Journal of Asian Natural Products Research Final purification of Frs 1 –6 was done on a Nova-pack HRC18 column (Waters C18 column, 78 mm £ 300 mm, 6 mm) to afford compound 4 (7.2 mg, tR: 96.7 min, MeOH – H2O 70:30 with 0.05% HOAc) from Fr. 1; compound 5 (15 mg, tR: 35.2 min, MeOH – H2O 69:31 with 0.1% HOAc) from Fr. 2; compound 9 (14 mg, tR: 16.6 min, MeOH – H2O 70:30 with 0.1% HOAc) from Fr. 3; compound 3 (30 mg, tR: 30.6 min, MeOH – H2O 70:30 with 0.1% HOAc) from Fr. 4; compound 2 (8 mg, tR: 44.8 min, MeOH – H2O 70:30 with 0.1% HOAc) and compound 1 (6.2 mg, tR: 53.2 min, MeOH – H2O 70:30 with 0.1% HOAc) from Fr. 5; and compound 8 (10 mg, tR: 12.8 min, MeOH –H2O 65:35 with 0.05% HOAc) from Fr. 6, respectively. Purification of Frs 7 and 8 was done on YMC-Pack ODS-A column (YMC C18 column, 30 mm £ 300 mm, 50 mm) to afford compound 7 (15 mg, tR: 16.5 min, MeOH – H2O 65:35 with 0.05% HOAc) and compound 6 (25 mg, tR: 26.3 min, MeOH – H2O 70:30 with 0.05% HOAc), respectively. 3.3.1 (3b)-22-Oxoolean-12-en-3-yl b-D -galactopyranosyl-(1 ! 2)-b-D glucopyranosiduronic acid (1) A white amorphous powder, ½a25 D þ 13.2 (c ¼ 1.40, MeOH); IR (KBr) nmax cm21: 3448, 2927, 1700, 1640, 1636, 1400, 1385, 1115, 1045. For 1H and 13C NMR spectral data, see Table 1. ESI-TOF-MS (negative) m/z: 411.4143 [M 2 C12H18O11 2 CO 2 H] – , 439.3599 [M – C12 H18O 11 – H] – , 597.3703 [M – C6H 10 O 5 – H 2O – H] – , 777.4423 [M – H] – (calcd for C42H65O13, 777.4425). 3.3.2 (3b)-22-Oxoolean-12-en-3-yl a-L -rhamnopyrannosyl-(1 ! 2)-b-D xylopyranosyl-(1 ! 2)-b-D glucopyranosiduronic acid (2) A white amorphous powder, ½a25 D 2 21.8 (c ¼ 2.00, MeOH); IR (KBr) nmax cm21:
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3430, 2927, 1710, 1645, 1635, 1399, 1385, 1184, 1127, 1045. For 1H and 13C NMR spectral data, see Table 2. ESI-TOF-MS (negative) m/z: 411.4149 [M – C17H26O14 – CO –H] – , 439.3628 [M –C17H26O14 – H] – , 597.3689 [M – C11H 18O 8 – H2 O – H] – , 747.4314 [M – C6H10O4 – H] – , 893.4897 [M – H] – (calcd for C47H73O16, 893.4899). 3.4 Acid hydrolysis of compounds 1 and 2 Each compound (2 mg) was heated in 3N HCl (dioxane – H2O, 1:1, 2 ml) at 808C for 4 h. After dioxane was removed, the reaction mixture was neutralized with 1 M NaOH and filtered. The filtrate was extracted with CHCl3 (2 £ 2 ml) to obtain the aglycone. By comparison on TLC with an authentic sample, the aglycone of 1 and 2 was determined to be abrisapogenol F (CHCl3 – MeOH, 20:1; Rf ¼ 0.65; reagent: EtOH –H2SO4, 90:10). The H2O layer was concentrated under reduced pressure to dryness, to give a residue of the sugar fraction. The crude sugar residue was analyzed by TLC in comparison with authentic samples (EtOAc – pyridine – anhydrous ethanol – H2 O, 8:1:1:2; Rf ¼ 0.57 (L -rhamnose), 0.41 (D -xylose), 0.24 (D -galactose), and 0.08 (D -glucuronic acid); reagent: aniline (2% in acetone) – diphenylamine (2% in acetone) – 85% H3PO4 (5:5:1). Acknowledgments This study was financially supported by the Research Fund for the Doctoral Program of Higher Education, China (No. 200801630007), and the 2008 Scientific Technology Plan Project from Science and Technology Department of Liaoning Province, China (No. 20082260233).
References [1] The Committee of Chinese Materia Medica, Chinese Materia Medica, Tomus 11 (Shanghai Press of Science and Technology, Shanghai, 1999), p. 619.
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[2] Y. Niiho, T. Yamazaki, Y. Nakajima, H. Itoh, T. Takeshita, J. Kinjo, and T. Nohara, Yakugaku Zasshi 109, 424 (1989). [3] C. Kim, S. Shin, H. Ha, and J.M. Kim, Arch. Pharm. Res. 26, 210 (2003). [4] Editorial Committee of China Pharmacopoeia, China Pharmacopoeia, Part I (China Chemical Industry Press, Beijing, 2010), p. 312. [5] Japanese Pharmacopoeia Editorial Committee, Japanese Pharmacopoeia, 15th ed. (Hirohawa Press, Tokyo, 2006), p. 1341. [6] J. Kinjo, T. Takeshita, Y. Abe, N. Terada, H. Yamashita, M. Yamasaki, K. Takeuchi, K. Murakami, T. Tomimatsu, and T. Nohara, Chem. Pharm. Bull. 36, 1174 (1988). [7] J. Kinjo, K. Aoki, M. Okawa, Y. Shii, T. Hirakawa, T. Nohara, Y. Nakajima, T. Yamazaki, T. Hosono, M. Someya, Y. Niiho, and T. Kurashige, Chem. Pharm. Bull. 47, 708 (1999). [8] D. Yuan, Y.Y. Xie, X. Bai, X. Wu, J.Y. Yang and C.F. Wu, J. Asian Nat. Prod. Res. 6, 471 (2009). [9] X. Chang, Y. Yuan, Y.Y. Xie, J.L. Hu, and D. Yuan, Chin. J. Med. Chem. 19, 241 (2009). [10] T. Kurihara and M. Kikuchi, Yakugaku Zasshi 95, 1283 (1975). [11] Y.O. Han, M.J. Han, S.H. Park, and D.H. Kim, J. Pharmacol. Sci. 93, 331 (2003). [12] H.U. Lee, E.A. Bae, and D.H. Kim, J. Pharmacol. Sci. 97, 541 (2005).
[13] H.I. Moon, J.C. Jung, and J. Lee, Bioorg. Med. Chem. 14, 7592 (2006). [14] K.Y. Park, G.O. Jung, J. Choi, K.T. Lee, and H.J. Park, Arch. Pharm. Res. 25, 320 (2002). [15] J.S. Park, M.S. Woo, D.H. Kim, J.W. Hyun, W.K. Kim, J.C. Lee, and H.S. Kim, J. Pharmacol. Exp. Ther. 320, 1237 (2007). [16] J. Choi, M.H. Shin, K.Y. Park, K.T. Lee, H.J. Jung, M.S. Lee, and H.J. Park, J. Med. Food 7, 31 (2004). [17] S.R. Oh, J. Kinjo, Y. Shii, T. Ikeda, T. Nohara, K.S. Ahn, J.H. Kim, and H.K. Lee, Planta Med. 66, 506 (2000). [18] Y. Niiho, Y. Nakajima, T. Yamazaki, M. Okamoto, R. Tsuchihashi, M. Kodera, J. Kinjo, and T. Nohara, J. Nat. Med. 64, 313 (2010). [19] M. Zeng, S.Q. Zheng, H.M. Zhang, and Z.W. Su, J. Pharm. Pract. 18, 344 (2000). [20] H. Miyao, Y. Sakai, T. Takeshita, J. Kinjo, and T. Nohara, Chem. Pharm. Bull. 44, 1222 (1996). [21] T. Takeshita, S. Hamada, and T. Nohara, Chem. Pharm. Bull. 37, 846 (1989). [22] Y. Ding, J. Kinjo, C.R. Yang, and T. Nohara, Chem. Pharm. Bull. 39, 496 (1991). [23] B. Cui, J. Inoue, T. Takeshita, J. Kinjo, and T. Nohara, Chem. Pharm. Bull. 40, 3330 (1992). [24] M. Udayama, J. Kinjo, and T. Nohara, Phytochemistry 48, 1233 (1998).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
New triterpenoid saponins from the flowers of Pueraria thomsonii a
b
a
a
a
Jing Lu , Jia-Hong Sun , Yao Tan , Yoshihiro Kano & Dan Yuan a
Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang, 110016, China b
Department of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 110016, China Published online: 29 Oct 2013.
To cite this article: Jing Lu, Jia-Hong Sun, Yao Tan, Yoshihiro Kano & Dan Yuan (2013) New triterpenoid saponins from the flowers of Pueraria thomsonii, Journal of Asian Natural Products Research, 15:10, 1065-1072, DOI: 10.1080/10286020.2013.802690 To link to this article: http://dx.doi.org/10.1080/10286020.2013.802690
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Journal of Asian Natural Products Research, 2013 Vol. 15, No. 10, 1065–1072, http://dx.doi.org/10.1080/10286020.2013.802690
New triterpenoid saponins from the flowers of Pueraria thomsonii Jing Lua, Jia-Hong Sunb, Yao Tana, Yoshihiro Kanoa and Dan Yuana* a
Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016, China; bDepartment of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
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(Received 4 March 2013; final version received 1 May 2013) Two new oleanane-type triterpenoid saponins, kakkasaponin II (1) and kakkasaponin III (2), were isolated from the methanol extract of the flowers of Pueraria thomsonii (Leguminosae), together with seven known oleanane-type triterpenoid saponins, phaseoside IV (3), sophoradiol monoglucuronide (4), kakkasaponin I (5), kaikasaponin III (6), soyasaponin I (7), soyasaponin III (8), and soyasaponin IV (9). The structures of 1 and 2 were elucidated by spectroscopic methods including IR, ESI-TOF-MS, and 1D and 2D NMR experiments. Keywords: Pueraria thomsonii; Leguminosae; triterpenoid saponins
1.
Introduction
Puerariae Flos, a folk herb, is used to treat the complication of diabetes and alcohol intoxication in China, Korea, and Japan [1– 3], and similarly, the roots of the plant are a well-known herbal medicine mentioned in Chinese and Japanese Pharmacopoeias [4,5]. Puerariae Flos is botanically from the flowers of Pueraria lobata (Willd.) Ohwi and Pueraria thomsonii Benth. (Leguminosae). Previous phytochemical studies on flowers of both Pueraria sp. reported the isolation of many saponins and isoflavones [6–10]. The isoflavones, such as kakkalide, tectoridin, and tectorigenin, possess hepatoprotective, hypoglycemic, hypolipidemic, antioxidant and antiinflammatory activities [11–15], meanwhile the saponins, such as kakkasaponin I, kaikasaponin III, and soyasaponin I, possess hepatoprotective, hypoglycemic, hypolipidemic, antioxidant, and anticomplementary activities [7,14,16,17]. The HPLC profile reported by Kinjo et al. and Niiho et al. [7,18] showed that the major saponins, kakkasaponin I, kaikasaponin III,
and soyasaponin I, are found in both P. lobata and P. thomsonii flowers. However, the minor saponins from both flowers have not yet been studied. In terms of resource utilization, further investigation on the saponins of P. thomsonii flowers is very important because of its richer botanical nature than that of P. lobata flowers [19]. This study describes the isolation and structural elucidation of two new oleananetype triterpenoid saponins (1 and 2) and seven known saponins (3 – 9) from P. thomsonii flowers. 2. Results and discussion The MeOH extract of P. thomsonii flowers was prepared by the reflux method. The EtOAc and n-BuOH fractions were obtained by partitioning the MeOH extract. Highperformance liquid chromotography diode array detection (HPLC-DAD) analysis (data not shown) indicated that the EtOAc fraction mainly contained tectoridin derivatives, while the n-BuOH fraction mainly contained saponins as well as minor tectoridin derivatives. Further isolation of
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
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the n-BuOH fraction led to two new compounds, kakkasaponin II (1) and kakkasaponin III (2), together with seven known triterpenoid saponins, phaseoside IV (3), sophoradiol monoglucuronide (4), kakkasaponin I (5), kaikasaponin III (6), soyasaponin I (7), soyasaponin III (8), and soyasaponin IV (9) (Figure 1). The structures of compounds 1 and 2 were elucidated by IR, ESI-TOF-MS, and 1D and 2D NMR techniques. Compounds 3–9 were identified by comparing their NMR and MS data with reported values [6,17,20–23], among which, compounds 3 and 9, or compounds 4 and 8 were for the first time isolated from Pueraria sp. or from P. thomsonii flowers, respectively. The inhibitory activities of the isolates on AR (aldose reductase) from bovine lens have been assayed. Nevertheless, all of them showed little activity (data not shown), and the bioactivities of triterpenoid saponins from P. thomsonii flowers need to be investigated in subsequent research. Compound 1 was isolated as an amorphous powder and showed positive results to the Molish and Lieberman–Burchard tests. The molecular formula was determined to be C42H66O13 from a pseudomolecular ion peak at m/z 777.4423 [M–H] – in the negative ESITOF-MS. Two important fragment ion peaks at m/z 597.4 [M–C6H10O5 –H2O–H] – and 439.4 [M–C12H18O11 –H] – suggested the
Figure 1. Structures of triterpenoid saponins 1 – 9.
presence of a glucuronosyl and a hexosyl group. Further loss of 28 amu [–CO] from it produced a fragment ion peak at m/z 411.4, suggesting the presence of a carbonyl group. The IR spectrum showed absorption bands due to a hydroxyl (3448 cm21), a carboxylic carbonyl (1700 cm21), and an alkenyl (1636 cm21). The 1H and 13C NMR spectra (Table 1) showed typical signals for an oleanene-type sapogenol, including eight methyl groups (dH 0.83, 0.85, 0.89, 0.93, 1.13, 1.16, 1.27, and 1.33, 3H each, s), an olefinic proton (dH 5.23, t, J ¼ 3.78 Hz), two olefinic carbons (dC 123.1 and 141.9), a carbonyl group (dC 215.7), and an oxygenated carbon (dC 89.1). The location of the double bond at C12 –C13 and the carbonyl group at C22 was confirmed by the HMBC correlations between H-27 and C-13 and between H-28 and C-22, respectively. The resonances for the carbons and protons located on the aglycone framework had a close resemblance to those of the known abrisapogenol F, and they were assigned according to the 1H and 13C NMR data as well as the 2D NMR data reported in the literature for abrisapogenol F [21]. The carbohydrate chain of 1 consisted of two monosaccharide residues, as deduced from the signals of two anomeric carbons (dC 105.4 and 107.3) in the 13C NMR spectrum. Both anomeric carbons were correlated with the corresponding signals of anomeric
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Table 1. NMR (600 MHz) data of compound 1 in C5D5N (d in ppm, J in Hz).
dC
dH
1
38.7 (t)
2
26.7 (t)
3 4 5 6 7
89.1 (d) 39.6 (s) 55.7 (d) 18.4 (t) 32.9 (t)
1.37 – 1.39 (m, Hax) 0.79 – 0.83 (m, Heq) 1.88 – 1.90 (m, Hax) 2.21 – 2.25 (m, Heq) 3.31 (dd, J ¼ 11.5, 4.6, 1H)
8 9 10 11 12 13 14 15 16
39.9 (s) 47.7 (d) 36.8 (s) 23.8 (t) 123.1 (d) 141.9 (s) 42.0 (s) 25.4 (t) 27.4 (t)
17 18 19
47.9 (s) 47.9 (d) 46.6 (t)
20 21
34.2 (s) 50.9 (t)
HMBCa
COSYb
H-3 C-10
Hax-2
0.72 (d, J ¼ 11.4, 1H) 1.47 – 1.49 (m, Heq), 1.28c 1.39 – 1.42 (m, Hax) 1.23 – 1.26 (m, Heq)
C-25
Hax-6 H-5
1.57 (t, J ¼ 9.6, 1H)
C-10
1.77 – 1.83 (m, 2H) 5.23 (br s) 1.63 – 1.66 (m, Hax), 0.97c 2.08 – 2.11 (m, Hax) 1.14c 2.35 (dd, J ¼ 14.8, 3.7, 1H) 2.14 – 2.17(m, Hax), 1.29c 2.11 – 2.15 (m, Hax) 2.58 (d, J ¼ 13.8, Heq)
C-18, C-20, C-30 C-13
Hax, Heq-19 H-18 H-18
C-19, C-20, C-30 C-20, C-22, C-29
22 23 24 25 26 27 28 29 30 Glu 10 20 30 40 50 60 Gal 100 200 300 400 500 600
215.7 (s) 28.2 (q) 16.8 (q) 15.7 (q) 16.9 (q) 25.6 (q) 21.0 (q) 31.9 (q) 25.3 (q) 105.4 (d) 84.0 (d) 77.5 (d) 74.8 (d) 77.7 (d) 172.9 (s) 107.3 (d) 73.2 (d) 75.0 (d) 69.5 (d) 77.0 (d) 61.3 (t)
1.33 (s, 3H) 1.13 (s, 3H) 0.85 (s, 3H) 0.89 (s, 3H) 1.27 (s, 3H) 1.16 (s, 3H) 0.93 (s, 3H) 0.83 (s, 3H) 5.04 (d, J ¼ 7.8, 1H) 4.31 – 3.35 (m, 1H) 4.62c 4.63 – 4.66 (m, 1H) 4.38 – 4.40 (m, 1H) n.d. 5.25 (d, J ¼ 7.8, 1H) 4.60c 4.20 – 4.22 (m, 1H) 4.73 – 4.74 (m, 1H) 4.08 – 4.10 (m, 1H) 4.42 – 4.44 (m, 1H) 4.67c
C-3, C-4, C-5, C-24 C-3, C-4, C-5, C-23 C-5, C-10 C-7, C-8, C-9, C-14 C-8, C-13, C-14 C-17, C-18, C-22 C-19, C-20, C-21 C-19, C-20, C-21 C-3 C-10 , C-30 C-40
C-20 C-300 C-600
H-20 H-10
H-200 H-100 , H-300 H-200 , H-400 H-300 H-600 H-500
Notes: All chemical shift assignments were carried out on the basis of HSQC and HMBC NMR techniques. n.d., not determined. a HMBC correlations, optimized for 7 Hz, are from proton(s) stated to the indicated carbon. b1 H– 1H COSY correlations. c Overlapped with other signals, chemical shift obtained from 2D correlation.
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protons (dH 5.04, d, J ¼ 7.8 Hz and 5.25, d, J ¼ 7.8 Hz) by the heteronuclear singlequantum coherence (HSQC) spectrum. The signals of a glucuronide and a galactose residue were also identified, while the coupling constants of the anomeric protons indicated b-configuration of both glycosidic bonds [20]. All sugars of glycosides from the leaves of P. lobata belong to the D -series, as established earlier [19]. The position of interglycosidic linkage was confirmed on the basis of the HMBC correlation between H-1 of the galactose and C-2 of the glucuronide residue (Figure 2). The linkage of the carbohydrate moiety to aglycone was determined by HMBC correlation between H-1 of the glucuronic acid residue and C-3 of the aglycone, as well as by the downfield shift of C-3 (þ10.2 ppm) and the upfield shift of H-3a (–1.21 ppm) relative to the corresponding signals of abrisapogenol F [21]. Acid hydrolysis of 1 with 3N HCl yielded glucuronic acid, galactose, and aglycone abrisapogenol F, which were identified by co-thin layer chromatography (TLC) with authentic samples. Therefore, the structure of 1 was determined to be abrisapogenol F 3-O-b-D -galactopyranosyl(1 ! 2)-b-D -glucuropyranoside, named as kakkasaponin II.
Compound 2 was isolated as a white amorphous powder and showed positive results to the Molish and Lieberman– Burchard tests. The molecular formula was determined to be C47H74O16 from a pseudomolecular ion peak at m/z 893.4897 [M–H] – in negative ESI-TOF-MS. Four important fragment ion peaks at m/z 747.4 [M–C6H10O4 –H] – , 597.4 [M–C11H18O8 – H2O – H] – , 439.4 [M – C17H26O14 – H] – , and 411.4 [M – C17H26O14 – CO – H] – suggested the presence of a hexosyl, a pentosyl, a glucuronosyl, and a carbonyl group. The 1H and 13C NMR data (Table 2) together with the aglycone ion peak at m/z 439.4 indicated that the aglycone part of 2 was the same as that of 1. The carbohydrate chain consisted of three monosaccharide residues, as deduced from the signals of three anomeric carbons (dC 102.4, 102.7, and 105.2). These signals were correlated with the corresponding signals of anomeric protons (dH 6.39, br s; 5.76, d, J ¼ 7.3 Hz and 4.94, d, J ¼ 7.8 Hz) by the HSQC spectrum. The chemical shifts and coupling constants of the anomeric protons indicated a b-glucuronosyl, a b-xylopyranosyl group, and the remaining anomeric carbon at dC 102.4 was determined as a for rhamnose residue, whose NMR data were similar to
Figure 2. Key HMBCs of the sugar portion of compounds 1 and 2.
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Table 2. NMR (600 MHz) data of compound 2 in C5D5N (d in ppm, J in Hz). dC
dH
1 2
38.8 (t) 26.5 (t)
3 4 5 6
89.9 39.7 55.7 18.4
(d) (s) (d) (t)
0.85–0.88 (m, Heq), 1.39c 1.81–1.85 (m, Hax) 2.14–2.17 (m, Heq) 3.23 (dd, J ¼ 10.6, 3.6, 1H)
7 8 9 10 11 12 13 14 15
32.9 39.9 47.8 36.8 23.8 123.8 141.9 42.0 25.4
(t) (s) (d) (s) (t) (d) (s) (s) (t)
16
27.4 (t)
17 18 19
47.9 (s) 47.9 (d) 46.6 (t)
20 21
34.2 (s) 50.9 (t)
22 23 24 25 26 27 28 29 30 Glu 10 20 30 40 50 60 Xyl 100 200 300 400 500
215.7 (s) 28.3 (q) 16.7 (q) 15.7 (q) 16.9 (q) 25.5 (q) 21.0 (q) 31.9 (q) 25.3 (q) 105.2 (d) 78.7 (d) 77.0 (d) 74.0 (d) 77.7 (d) n.d. 102.7 (d) 79.3 (d) 78.1 (d) 71.0 (d) 66.8 (t)
Rha 1000 2000 3000 4000 5000 6000
102.4 72.4 72.6 74.3 69.6 19.0
(d) (d) (d) (d) (d) (q)
0.76 (d, J ¼ 12.0, 1H) 1.52–1.54 (m, Heq) 1.31c 1.23–1.25 (m, Heq), n.d.
HMBCa
COSYb H-3 H-3 Hax, Heq-2
C-25
Hax-6 H- 5
1.62 (t, J ¼ 8.4, 1H) 1.85–1.90 (m, 2H) 5.24c 1.66–1.69 (m, Hax) 0.97–0.99 (m, Heq) 2.06–2.08 (m, Hax) 1.11c
Hax-16
2.36 (dd, J ¼ 12.0, 3.8, 1H) 2.12–2.14 (m, Hax) 1.29c
C-13
Hax, Heq-19 H-18 H-18
2.08–2.12 (m, Hax) 2.58 (d, J ¼ 13.8, Heq)
C-19, C-20, C-30 C-22, C-29
1.33 (s, 3H) 1.08 (s, 3H) 0.84 (s, 3H) 0.90 (s, 3H) 1.28 (s, 3H) 1.16 (s, 3H) 0.94 (s, 3H) 0.83 (s, 3H) 4.94 (d, J ¼ 7.8, 1H) 4.56c n.d. 4.36–4.39 (m, 1H) 4.28–4.31 (m, 1H) n.d. 5.76 (d, J ¼ 7.3, 1H) 4.15–4.17 (m, 1H) 4.36–4.39 (m, 1H) 4.17–4.21 (m, 1H) 3.53–3.56 (m, 1H) 4.29–4.33 (m, 1H) 6.39 (br s, 1H) 4.77c 4.70c 4.33–4.36 (m, 1H) 5.07c 1.77 (d, J ¼ 6.0, 3H)
C-3, C-4, C-5, C-24 C-3, C-4, C-5, C-23 C-5, C-10 C-7, C-8, C-9, C-14 C-8, C-13, C-14 C-17, C-18, C-22 C-19, C-20, C-21 C-19, C-20, C-21 C-3
Hax-15
H-40 H-20 C-20
C-400 C-200 , C-2000 , C-5000
C-4000 , C-5000
H-500 H-500 H-400 H-100 H-2000 H-1000 H-4000 H-3000 , H-5000 H-4000 , H-6000 H-5000
Note: All chemical shift assignments were carried out on the basis of HSQC and HMBC NMR techniques. n.d., not determined. a HMBC correlations, optimized for 7 Hz, are from proton(s) stated to the indicated carbon. b H – 1H COSY correlations. c Overlapped with other signals, chemical shift obtained from 2D correlation.
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those observed in baptisiasaponin I isolated from the roots of Baptisia australis (Leguminosae) earlier [24]. The interglycosidic linkages were confirmed by the HMBC correlations between H-1 of the xlyose and C-2 of the glucuronide residue and between H-1 of the rhamnose and C-2 of the xylose residue (Figure 2). The linkage of the carbohydrate moiety to aglycone was determined by HMBC correlation between H-1 of the glucuronic acid residue and C-3 of the aglycone, as well as by the downfield shift of C-3 (þ11.0 ppm) and the upfield shift of H-3a (–1.27 ppm) relative to the corresponding signals of abrisapogenol F [21]. Acid hydrolysis of 2 with 3N HCl yielded glucuronic acid, xlyose, rhamnose residues (identified by co-TLC with authentic samples) as well as the aglycone abrisapogenol F. Therefore, the structure of 2 was determined to be abrisapogenol F 3O-a-L -rhamnopyrannosyl-(1 ! 2)-b-D xylopyranosyl-(1 ! 2)-b-D -glucuropyranoside, named as kakkasaponin III. 3. 3.1
Experimental General experimental procedures
Optical rotations were recorded on a Perkin-Elmer 241MC automatic polarimeter (Perkin-Elmer, Waltham, MA, USA). IR spectra were obtained on a Bruker IFS-55 infrared spectrometer (Bruker, Faellanden, Switzerland). 1D and 2D NMR spectra were recorded separately on a Bruker ARX-600 or ARX-300 spectrometer (Bruker). ESI-TOF-MS were acquired on a Micro TOF Bruker Daltonics mass spectrometer with a resolution of 25,000 (10% Valley, Bruker, Karlsruhe, Germany) and ESI-MS were acquired on a Shimadzu QP8000a mass spectrometer (Shimadzu Corporation, Shimadzu, Japan). Preparative HPLC was carried out using a Shimadzu’s LC-6A or Shimadzu’s LC-8A solvent delivery pump and Shimadzu’s SPD-10AVP detector (Shimadzu Corporation). Column chromatography (CC) was carried out on
silica gel (200 – 300 mesh, Qingdao Haiyang Chemical Group Co., Qingdao, China), Sephadex LH-20 (GE Healthcare, Uppsala, Sweden), octadecylsilyl silica (ODS) (YMC Co., Ltd, Kyoto, Japan), MDS-5 reversed-phase packing (200 – 300 mesh, Beijing Medicine Technology Center, Beijing, China), and Waters C18 column (7.8 mm £ 250 mm, 6 mm, Waters Co., Ltd, Milford, MA, USA). TLC was carried out on Merck silica gel 60F254 plates (E. Merck, Darmstadt, Germany). Other chemical reagents were of analysis or HPLC grades. Double distilled water was used in this study. The authentic sugars were purchased from SigmaAldrich (St Louis, MO, USA). 3.2 Plant material The flowers of P. thomsonii were collected in October 2006 in Hengyang, Hunan Province, China. Species identification was confirmed by Prof. Wei-Ning Wang, Liaoning Institute for Food and Drug Control, China. A voucher specimen (A0270610F) has been deposited in the Department of Traditional Chinese Medicines, Shenyang Pharmaceutical University. 3.3 Extraction and isolation The air-dried flowers (7.0 kg) of P. thomsonii were refluxed with MeOH (2 £ 42 liters; each 3 h) and evaporated in vacuo. The MeOH extract (1030 g) was partitioned between hexane (3 £ 3 liters) and H2O (3 £ 3 liters), and the H2O phase was extracted with EtOAc (3 £ 3 liters) to give an EtOAc fraction. The H2O phase was further extracted with n-BuOH (3 £ 3 liters) to give an n-BuOH fraction. The dried n-BuOH fraction was subjected to CC (silica gel, 1.5 kg), gradient of CHCl3 – MeOH – H2 O, 70:25:3 ! 60:40:10) to give Frs 1 – 8. These fractions were further subjected to repeated CC (MDS-5, gradient of MeOH –H2O, 50:50 ! 90:10; ODS, gradient of MeOH – H2O, 60:40 ! 80:20).
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Journal of Asian Natural Products Research Final purification of Frs 1 –6 was done on a Nova-pack HRC18 column (Waters C18 column, 78 mm £ 300 mm, 6 mm) to afford compound 4 (7.2 mg, tR: 96.7 min, MeOH – H2O 70:30 with 0.05% HOAc) from Fr. 1; compound 5 (15 mg, tR: 35.2 min, MeOH – H2O 69:31 with 0.1% HOAc) from Fr. 2; compound 9 (14 mg, tR: 16.6 min, MeOH – H2O 70:30 with 0.1% HOAc) from Fr. 3; compound 3 (30 mg, tR: 30.6 min, MeOH – H2O 70:30 with 0.1% HOAc) from Fr. 4; compound 2 (8 mg, tR: 44.8 min, MeOH – H2O 70:30 with 0.1% HOAc) and compound 1 (6.2 mg, tR: 53.2 min, MeOH – H2O 70:30 with 0.1% HOAc) from Fr. 5; and compound 8 (10 mg, tR: 12.8 min, MeOH –H2O 65:35 with 0.05% HOAc) from Fr. 6, respectively. Purification of Frs 7 and 8 was done on YMC-Pack ODS-A column (YMC C18 column, 30 mm £ 300 mm, 50 mm) to afford compound 7 (15 mg, tR: 16.5 min, MeOH – H2O 65:35 with 0.05% HOAc) and compound 6 (25 mg, tR: 26.3 min, MeOH – H2O 70:30 with 0.05% HOAc), respectively. 3.3.1 (3b)-22-Oxoolean-12-en-3-yl b-D -galactopyranosyl-(1 ! 2)-b-D glucopyranosiduronic acid (1) A white amorphous powder, ½a25 D þ 13.2 (c ¼ 1.40, MeOH); IR (KBr) nmax cm21: 3448, 2927, 1700, 1640, 1636, 1400, 1385, 1115, 1045. For 1H and 13C NMR spectral data, see Table 1. ESI-TOF-MS (negative) m/z: 411.4143 [M 2 C12H18O11 2 CO 2 H] – , 439.3599 [M – C12 H18O 11 – H] – , 597.3703 [M – C6H 10 O 5 – H 2O – H] – , 777.4423 [M – H] – (calcd for C42H65O13, 777.4425). 3.3.2 (3b)-22-Oxoolean-12-en-3-yl a-L -rhamnopyrannosyl-(1 ! 2)-b-D xylopyranosyl-(1 ! 2)-b-D glucopyranosiduronic acid (2) A white amorphous powder, ½a25 D 2 21.8 (c ¼ 2.00, MeOH); IR (KBr) nmax cm21:
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3430, 2927, 1710, 1645, 1635, 1399, 1385, 1184, 1127, 1045. For 1H and 13C NMR spectral data, see Table 2. ESI-TOF-MS (negative) m/z: 411.4149 [M – C17H26O14 – CO –H] – , 439.3628 [M –C17H26O14 – H] – , 597.3689 [M – C11H 18O 8 – H2 O – H] – , 747.4314 [M – C6H10O4 – H] – , 893.4897 [M – H] – (calcd for C47H73O16, 893.4899). 3.4 Acid hydrolysis of compounds 1 and 2 Each compound (2 mg) was heated in 3N HCl (dioxane – H2O, 1:1, 2 ml) at 808C for 4 h. After dioxane was removed, the reaction mixture was neutralized with 1 M NaOH and filtered. The filtrate was extracted with CHCl3 (2 £ 2 ml) to obtain the aglycone. By comparison on TLC with an authentic sample, the aglycone of 1 and 2 was determined to be abrisapogenol F (CHCl3 – MeOH, 20:1; Rf ¼ 0.65; reagent: EtOH –H2SO4, 90:10). The H2O layer was concentrated under reduced pressure to dryness, to give a residue of the sugar fraction. The crude sugar residue was analyzed by TLC in comparison with authentic samples (EtOAc – pyridine – anhydrous ethanol – H2 O, 8:1:1:2; Rf ¼ 0.57 (L -rhamnose), 0.41 (D -xylose), 0.24 (D -galactose), and 0.08 (D -glucuronic acid); reagent: aniline (2% in acetone) – diphenylamine (2% in acetone) – 85% H3PO4 (5:5:1). Acknowledgments This study was financially supported by the Research Fund for the Doctoral Program of Higher Education, China (No. 200801630007), and the 2008 Scientific Technology Plan Project from Science and Technology Department of Liaoning Province, China (No. 20082260233).
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