Glyceroglycolipids from Serratula strangulata

Jing Qiu Daia,*, Qi Xiu Zhua, Chen Yang Zhaob, Li Yanga, Yu Lia

aNational Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, Gansu, People’s Republic of ChinabDepartment of Biology, Lanzhou University, Lanzhou 730000 Gansu, People’s Republic of China

Received 30 November 2000; received in revised form 17 May 2001

Abstract

The rhizomes of Serratula strangulata yielded three glyceroglycolipids, i.e. 1,2-di-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-(6-amine-6-deoxy-a-d-glucosyl)-glycerol, 1,2-di-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-(6-p-hydroxy-phenyl-propionamido-6-deoxy-a-d-glucosyl)-glycerol and 1,2-di-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-[a-d-glucose(1-6)-b-d-allose]-glycerol, as well as a known sesqui-terpene lactone and three known phytoecdysones. Their structures were elucidated on the basis of spectral data, especially by 2D NMR

spectroscopic methods and chemical conversion. These compounds exhibited significant antibacterial and antitumor activities.# 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Serratula strangulata; Compositae; Glyceroglycolipids; Cytotoxicity; Antimicrobial

1. Introduction

Serratula strangulata (Compositae) is a perennial her-baceous plant growing mainly in North China. Its rhi-zomes have been used as a traditional chinese medicinefor treatment of chickenpox, toxicosis, and high choles-terol levels since ancient times (Liu, 1987). Its phyto-chemical or pharmacological properties have not beenreported to date. In our effort to find active componentsfrom this plant, we found three new glyceroglycolipids, i.e.1,2-di-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-(6-amine-6-deoxy-a-d-glucosyl)-glycerol (1), 1,2-di-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-(6-p-hydroxy-phenyl-propiona-mido-6-deoxy-a-d-glucosyl)-glycerol (2) and 1,2-di-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-[a-d-glucose(1-6)-b-d-allose]-glycerol (3), as well as four known compounds,centaurepensin (4), 20-hydroxyecdysone (5), 25-deoxy-11,20-dihydroxyecdysone (6) and 20-hydroxyecdysone-20,22-monoacetonide (7) from the alcohol extract of thewhole plant. We report herein the isolation, structuralelucidation and antibacterial and antitumor activities ofthe above three new glyceroglycolipids. It was found thatthese compounds exhibited significant antibacterial andantitumor activities, especially 2 possess strong cytotoxi-city for B 16 (mouse melanoma).

2. Results and discussion

From the EtOAc- and BuOH-soluble fraction of anEtOH extract of the rhizome of S. strangulata, sevencompounds (1–7) were purified by repeated chromato-graphy on silica gel. Each of the isolates was subjectedto detailed spectroscopic analyses in order to establishtheir chemical structures.Compound 1 was isolated as a colorless gum. FAB–

MS which gave a quasi-molecular ion peak at m/z 796(M+Na)+, 780 (M+Li)+, as well as its elemental ana-lysis (found: C 69.85%, H 9.70%, N 1.80%; required: C69.86%, H 9.70%, N 1.81%) indicated that the mole-cular formula should be C45H75NO9, which was provedby HR–FAB–MS (m/z 773.5422, calc. 773.5441). Its IRspectrum showed strong absorptions for hydroxyl andester groups (3464 cm�1, 1729 cm�1), while the 1H NMRspectrum exhibited two terminal methyl signals (� 0.90,6H, t), a broad methylene signal at 1.23 ppm which theintegration showed 20 protons, and the signal (� 2.30,4H, m) due to two methylene protons linked to a car-bonyl function. The 1H NMR spectrum of 1 furtherrevealed olefinic proton (� 5.30, 12H, m) and methyleneprotons linked to double bonds (� 1.69, 4H; � 2.01, 4H; �2.75, 8H). These were consistent with the proton signalsof two linolenic acid moieties (Pchelkin and Ver-eshchagin, 1980). In addition, coupling constant analysisof the homonuclear decoupling spectrum defined the

0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.

PI I : S0031-9422(01 )00308-9

Phytochemistry 58 (2001) 1305–1309

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* Corresponding author.

E-mail address: daij1@21cn.edu.cn (J.Q. Dai).

sugar component as glucose; this being consistent withthe 13C spectrum (Table 1). The configuration at C-10 ofthe glucose was determined to be a by the couplingconstant of the anomeric proton (J=3.5 Hz). Treatmentof 1 with NaOMe–MeOH gave mono-glucosyl-glyceroland linolenic acid. The 13CNMR and DEPT spectrum of1 confirmed the presence of linolenic acid (Frangulyan,1987) and an a-aminoglucose moieties. Simultaneously,they also revealed three carbon signals connected tooxygen atoms: �C 62.62 (CH2), 69.73 (CH) and 64.63(CH2), which pointed to the presence of a glyceryl moi-ety. The sites of attachment of the two linolenic acidsand aminoglucose moiety of 1 were determined to be at

C-1, C-2, C-3 respectively, by means of the HMBC,1H-1HCOSY and HMQC spectrum. Because the upfieldshift of C-60 in the aminoglucose observed of 1H NMRand 13C NMR spectral analysis, the amine group shouldbe attached to C-60. Thus, 1 was assigned as 1,2-di-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-(6-amine-6-deoxy-a-d-glucosyl)-glycerol. This appears to be the first reportedoccurrence of glucosaminediacylglycerol in the genusSerratula.Compound 2 was isolated as a colorless gum. FAB–

MS showed [M+Na]+ at m/z 944 and [M+Li]+ at 928.Its 1H, 13C and DEPT spectrum were similar to those of1. In the 1H, 13C NMR spectral analyses of 2, the extra

1306 J.Q. Dai et al. / Phytochemistry 58 (2001) 1305–1309

signals at �H 7.02 (dd, J=7.2, 2.1 Hz), 6.69 (dd, J=7.2,2.1 Hz) and �C 40.12 (CH2), 32.22 (CH2), 172.48 (esterC¼O) revealed the presence of a p-substituted benzenering and the partial structure –CH2–CH2– and carbonylgroup. 1H–1H COSY and HMQC spectrum confirmedabove assignments. According to the results of HMBCspectrum: C-100/H-60, H-200, H-300; C-400/H-300, H-500 (900); C-700/H-600 (800) (Fig. 1), the structure of 2was deduced as 1,2-di-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-(6-p-hydroxy-phenyl-propionamido-6-deoxy-a-d-glucosyl)-glycerol.Compound 3 was obtained as a colorless gum. The IR

spectrum showed absorption bands due to hydroxyl andester carbonyl at 3460, 1729 cm�1. The molecular for-mula C51H84O15 was established on the basis of thestrong quasi-molecular ion peak at m/z 959 [M+Na]+

and 943 [M+Li]+ in the FAB–MS, together with thesupport of combined spectroscopic methods (1H NMR,13C NMR and DEPT). The 1H and 13C NMR spectrumshowed that 3 had the same skeleton and type as 1 and2. Detailed analysis of the remaining signals in the 1Hand 13C NMR spectrum of 3 suggested that it possessedan allose moiety attached to the inner glucose unit. Onacid hydrolysis with 2 mol/l HCl, 3 gave glucose and

allose, and on acid hydrolysis with 0.5 mol/l HCl, 3

afforded only allose (identified by 13C NMR and TLC),confirming the above deduction. The b-configuration ofthe glucosidic bond between the glucose and allose resi-due was determined on the basis of anomeric carbonsignal at 103.2 ppm. The observation of the downfieldshift of the C-60 carbon confirmed a1-6 linkage of the twoglucide units (Gong, 1983). Thus, the structure of 3 wasassigned as 1,2-di-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-[a-d-glucose (1-6)-b-d-allose]-glycerol, which was furthersupported by HMBC and HMQC analysis.During the course of the isolation of the plant extract, a

sesquiterpene lactone and three phytoecdysones were alsoobtained in pure form. This appears to be the first repor-ted occurrence of sesquiterpenes in the genus Serratula.Each of these isolates was characterized by its spectralproperties and subsequently identified as centaurepensin(Harley-Mason et al., 1972), 20-hydroxyecdysone (Nishi-moto et al., 1987), 25-deoxy-11,20-dihydroxyecdysone(Zhang et al., 1992) and 20-hydroxyecdysone-20, 22-monoacetonide (Zhang et al., 1992) by comparison withpublished spectral data and/or reference samples.Previous studies have reported the cytotoxicity of

centaurepensin against SMMC-7721 cells (Syrov et al.,1991), and interesting pharmacological effects of ecdys-teroids on mammals including a stimulation of proteinsynthesis, and a reduction of blood glucose and choles-terol levels (Camps, 1991; Simon and Koolman, 1989).Murakami et al. (1990) also reported anti algal activityof unsaturated fatty acids. In our study, compound 1–3exhibited antibacterial activity against Bacillus. subtilis,Escherichia. coli and Staphylococcus. aureus. The resultswere compared with chloramphenicol and are summar-ized in Table 2. Meanwhile, the inhibitory effects ofcompounds 1–3 on the survival of three tumor cell lines,human hepatoma cells (SMMC-7721), human uterinecervix carcinoma cells (HeLa) and mouse melanoticcarcinoma cells (B16), were studied (Table 3). The sur-vival rates of cells were determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]method. 2 exhibited the most effective antitumor activ-ity, especially on B16, and 3 had no effect on survival ofthe three tested tumor cells even when its concentrationswas as high as 455 mg /ml.

Table 2

Antibacterial activity of compound 1, 2, 3a

B. subtilis E. coli S. aureus

Compound 1 + +++ +

Compound 2 ++ +++ +

Compound 3 ++ ++ +

Chloramphenicol +++ +++ +++

a Antimicrobial activity is defined as follows: +++=the diameter

is equal to 16–20 mm; ++=13–15 mm; +=10–12 mm.Fig. 1. The key correlations of 2 in HMBC (C*H).

Table 113C NMR data for 1, 2 and 3 (100.62 MHz, DMSO-d6, TMS)a

Carbon 1 DEPT 2 DEPT 3 DEPT

Sn-1 62.62 CH2 62.62 CH2 62.46 CH2

Sn-2 69.73 CH 69.71 CH 69.65 CH

Sn-3 64.63 CH2 64.14 CH2 66.42 CH2

a-Aminoglu a-Aminoglu a-Glu

10 98.29 CH 98.31 CH 99.59 CH

20 71.56 CH 71.61 CH 71.29 CH

30 72.86 CH 72.92 CH 72.94 CH

40 74.13 CH 74.16 CH 69.76 CH

50 68.53 CH 68.53 CH 71.47 CH

60 54.38 CH2 54.88 CH2 66.67 CH2

Amido moiety b-Allo

100 172.48 C 103.20 CH

200 40.38 CH2 70.68 CH

300 32.21 CH2 70.35 CH

400 129.54 C 66.35 CH

500, 900 127.68 CH 73.18 CH

600, 800 115.34 CH 60.63 CH2

700 156.17 C

a Assignment from 1H–1H COSY, HMQC and HMBC.

J.Q. Dai et al. / Phytochemistry 58 (2001) 1305–1309 1307

3. Experimental

3.1. General

FAB–MS were determined on a MS 50 (A.E.I. Brun-ner) and a ZAB–HS mass spectrometer. 1H, 13C and 2DNMR spectra were recorded on Bruker AM-400 MHzspectrometer using TMS as internal standard. Deuter-ated solvents (DMSO-d6 for 1–3 and CD3COCD3 for 4–7) were used. IR spectra were obtained as KBr pelletson Nicolet-5DX. IR spectrometer.

3.2. Plant material

The whole plants of S. strangulata were collected inZhuanglang county, Gansu Province of China in August1996 and were identified by Professor Yong-hong Zhangof Lanzhou University. A voucher specimen (No. 9602)has been deposited at the Lab. of Natural Products,Department of Chemistry, Lanzhou University, Lanz-hou, PR China.

3.3. Extraction and isolation

Air-dried and powdered whole plants of S. strangu-lata (3 kg) were exhaustively extracted with 95%EtOH atroom temperature. The extract was concentrated underreduced pressure. The residue (120 g) was suspended inH2O, extracted with petroleum ether, EtOAC and BuOH,respectively. The EtOAC extract (40 g) was subjected tocolumn chromatography on silica gel eluting with petrolandMe2COmixtures to afford 4 (50 mg), 5 (45 mg), 6 (28mg), 7 (16 mg). From the BuOH extract (60 g), threeglyceroglycolipids 1 (40 mg), 2 (20 mg), 3 (48 mg) werepurified by silica gel chromatography using CHCl3–MeOH mixtures as solvent.

3.4. 1,2-Di-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-(6-amine-6-deoxy-�-d-glucosyl)-glycerol (1)

Colorless gum. a½ �24D +25.5 (c 3.01, MeOH), FAB–

MS m/z: 796 (M+Na)+, 780 (M+Li)+; HR–FAB–MSm/z 773.5422, calc. for C45H75NO9 773.5441; Elementalanalysis found: C 69.85%, H 9.70%, N 1.80%; required:C 69.86%, H 9.70%, N 1.81%. �KBr

max 3464�3271, 2926,2253, 2126, 1658, 1120, 1026, 825. 1H NMR (DMSO-d6)

� 0.90 (6H, t, J=7.5 Hz), 1.23 (20H, m), 1.69 (4H, m),2.01 (4H, m), 2.30 (4H, m), 2.75 (8H, m), 2.54 (1H, dd,J=14.0, 7.0 Hz, H-60 a), 2.89 (1H, dd, J=14.0, 4.2 Hz,H-60 b), 2.90 (1H, dd, J=9.0, 9.1 Hz, H-40), 3.17 (1H,dd, J=9.6, 3.5 Hz, H-20), 3.34 (1H, dd, J=9.1, 9.6 Hz,H-30), 3.44 (1H, dd, J=10.4, 4.7 Hz, sn-3-Ha), 3.76 (1H,ddd, J=9.0, 7.0, 4.2 Hz, H-50), 3.87 (1H, dd, J=10.4, 5.9Hz, sn-3-Hb), 4.11 (1H, dd, J=10.0, 7.4 Hz, sn-1-Ha),4.32 (1H, dd, J=10.0, 2.1 Hz, sn-1-Hb), 4.56 (1H, d,J=3.5 Hz, H-10), 5.11 (1H, m, sn-2-H), 5.30 (12H, m).For 13C NMR spectral data, see Table 1.

3.5. 1,2-Di-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-(6-p-hydroxy-phenyl-propionamido-6-deoxy-�-d-glucosyl)-glycerol (2)

Colorless gum. a½ �24D +25.8 (c 2.7, MeOH); IR�KBr

max

cm�1: 3464, 3271, 2926, 2254, 2125, 1729, 1658, 1515,1458, 1371, 1028, 762, 627; FAB–MS m/z: 922 (M+H)+; HR–FAB–MS m/z 921.5946, calc. for C54H83

NO11 921.5969; 1H NMR (DMSO-d6) � 0.88 (6H, t,J=7.5 Hz), 1.15 (20H, m), 1.47 (4H, m), 1.99 (4H, m),1.21 (2H, m, H-300), 2.22 (4H, m), 2.23 (2H, m, H-200),2.73 (1H, dd, J=14.0, 7.0 Hz, H-60 a), 2.75 (8H, m),2.90 (1H, dd, J=14.0, 4.2 Hz, H-60 b), 2.93 (1H, dd,J=9.0, 9.1 Hz, H-40), 3.17 (1H, dd, J=9.6, 3.5 Hz, H-20), 3.33 (1H, dd, J=9.1, 9.6 Hz, H-30), 3.44 (1H, dd,J=10.4, 4.7 Hz, sn-3-Ha), 3.77 (1H, ddd, J=9.0, 7.0, 4.2Hz, H-50), 3.87 (1H, dd, J=10.4, 5.9 Hz, sn-3-Hb), 4.11(1H, dd, J=10.0, 7.4 Hz, sn-1-Ha), 4.32 (1H, dd,J=10.0, 2.1 Hz, sn-1-Hb), 4.56 (1H, d, J=3.5 Hz, H-10),5.11 (1H, m, sn-2-H), 5.30 (12H, m), 6.68 (2H, dd,J=2.1, 7.2 Hz, H-600, 800), 7.02 (2H, dd, J=2.1, 7.2 Hz,H-500, 900), For 13C NMR spectral data, see Table 1.

3.6. 1,2-Di-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-[�-d-glucose(1-6)-�-d-allose]-glycerol (3)

Colorless gum. a½ �24D +25.3 (c 6.01, MeOH). IR�KBr

max

cm�1: 3460, 1720, 1651; FAB–MS m/z: 959 (M+Na)+,937 (M+Li)+. HR–FAB–MS m/z 936.5811, calc. forC51H84O15 936.5810. 1H NMR (DMSO-d6) � 0.89 (6H,t, J=7.3 Hz), 1.20 (20H, m), 1.46 (4H, m), 1.97 (4H, m),2.22 (4H, m), 2.72 (8H, m), 3.78 (1H, dd, J=11.0, 5.3Hz, sn-3-Ha), 3.60 (1H, dd, J=11.0, 4.5 Hz, sn-3-Hb),4.22 (1H, dd, J=12.1, 7.0 Hz, sn-1-Ha), 4.43 (1H, dd,J=12.1, 2.9 Hz, sn-1-Hb), 4.66 (1H, dd, J=3.5 Hz, H-100), 5.08 (1H, m, sn-2-H), 5.26 (12H, m). For 13C NMRspectral data, see Table 1.

3.7. Alkaline treatment of 1

A solution of 1 (5.0 mg) in dry MeOH (1.0 ml) wastreated with 5% NaOMe–MeOH (0.2 ml) at room tem-perature for 10 min. The reaction mixture was neu-tralized by using ion exchange resin (Dowex 50 W�8)

Table 3

Antitumour activity of compound 1 and 2a

SMMC-7721 B16 Hela

Compound 1 351.4�6.1 157.3�2.5 168.2�1.9

Compound 2 151.6�6.3 70.3�2.2 121.9�3.1

Vincristine 63.2�1.8 70.7�2.8 67.2�2.2

a Activities are expressed as IC50 (50% inhibitory concentration) in

mg ml�1.

1308 J.Q. Dai et al. / Phytochemistry 58 (2001) 1305–1309

and the resin was removed by filtration. The filtrate wasextracted with hexane and the hexane layer was con-centrated under reduced pressure to yield methyl linole-nate (3.5 mg). The methyl ester was identified by GLCcomparison with authentic samples. Removal of the sol-vent from theMeOH layer under reduced pressure gave aresidue, which was purified by SiO2 column chromato-graphy (CHCl3–MeOH–H2O=6:4:1) to furnish mono-glucosyl-glycerol (1.8 mg).

3.8. Acid hydrolysis of 3

A solution of 3 (15 mg) in 0.5 M HCl (5 ml) washeated at 70 C for 3 h. After cooling, the reactionmixture was neutralized with 1% NaOH, and extractedwith CHCl3 (5 ml). In the aqueous layer, allose wasdetected with PC by direct comparison with theauthentic sample of D-allose, and 13C NMR of theproduct was consistent with those of authentic sample.

3.9. Antimicrobial assays

Three strains of bacteria, B. subtilis, E. coli and S.aureus, were cultured under agar. The paper-diskmethod (Xu and Bian, 1982) was used as an anti-microbial test, 0.1 ml of 100 mg/ml of compounds 1, 2, 3or chloramphenicol (used as positive control) was addedto each piece of paper. After 1 h, the disks were driedand placed on to a culture dish at 37 C for 24 h. Theantimicrobial activity was calculated by the diameter (inmillimeters) of the antibacterial circle. Each test wasperformed in duplicate.

3.10. Cytotoxicity assays

The cytotoxicity of compounds 1–3 was tested inthree cell lines: SMMC-7721 (human hepatoma), B16(mouse melanoma) and HeLa (human carcinoma ofuterine cervix). Cells were cultured at 37 C under ahumidified atmosphere of 5% CO2 in RPMI 1640 med-ium supplemented with 10% fetal calf serum and dis-persed in replicate 96-well plates with 1�104 cells/well for24 h. Compounds 1–3 (10–400 mM) or vincristine (used asa positive control) were then added. After 48 h exposureto the toxins, cell viability was determined by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]

(MTT) colorimetric assay (Price and McMillan, 1990)by measuring the absorbance at 595 nm with an ESILAreader. Each test was performed in triplicate.

Acknowledgements

The authors are grateful to Professor Yong-hongZhang, Department of Chemistry, Lanzhou University,for her help in identification of plant material, and theNational Laboratory of Applied Organic Chemistry andAnalysis Center, Lanzhou University, PR China formeasuring 1H,13C NMR spectra and FAB–MS, HR–FAB–MS, respectively. This research project was sup-ported by the Natural Science Foundation of GansuProvince in China (ZS001-A25-001-Z).

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