B. Wu, W. H. Lin1, H. Y. Gao,l. Zheng,l. J. Wu* and C. S. Kim2

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, China-110 016, 1School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, China-110 016, 2Faculty of Agriculture, Kochi University, Nankoku, Japan-783-8502.

*Corresponding Author:
J. Wu
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, China-110 016
E-mail: wulijun_111@hotmail.com
Date of Submission 4 June 2005
Date of Revision 23 August 2005
Date of Acceptance 20 May 2006
Indian J Pharm Sci, 2006, 68 (3) 332-336  

DOI: 10.4103/0250-474X.26668

Abstract

Fractionation of a butanol extract of Senecio cannabifolius Less. led to the isolation of two novel monoterpenoid derivatives, named cannabiside D and cannabiside E, whose structures were determined by spectroscopic analyses as 1-(2-hydroxy-2,6,6-trimethyl-4-β-D- glucosyloxy-cyclohexylidene)-butane-2, 3-dione, 6-Hydroxy-3-(3-O-β-D-glucopyranosyl- but-1-enyl)-2, 4,4-trimethyl-cyclohex-2-enone, along with two known glycosides. All the compounds had antibacterial activity, showing particularly potent activity against Staphylococcus aureus IFO 3060 and Bacillus subtilis.

The plant Senecio cannabifolius Less. is a member of the family Compositae and distributes mainly in the Northeast and Hebei Province of China, Korea, Japan, and far east of pre-Soviet Union. It is used as a traditional remedy for treating virus influenza, enteritis, and pneumonia in China [1,2]. Plants belonging to the genus are notable for producing a wide variety of pyrrolizidine alkaloids and furoeromophilanes. The large, very diverse genus Senecio has already been studied extensively for its secondary chemicals [3]. In this paper, we report the isolation and structure elucidation of the four monoterpenoid derivatives isolated from the butanol extract of the herba of Senecio cannabifolius Less., along with their antibacterial activity.

Materials and Methods

1H- and 13C-NMR, DEPT, COSY, HMQC and HMBC spectra were measured using JNM-LA400 (JEOL Datum Ltd., Japan). Chemical shifts are shown in δ (ppm) with tetramethylsilance (TMS) used as an internal reference. UV spectra were recorded on an UV-VIS recording spectrophotometer (Shimadzu UV-2201), and IR spectra on a Bruker IFS 55 (Switzerland). HR-ESI-MS (positive ion-mode) was obtained with Finnigan LCQ (USA) using a heated capillary temperature of 180°, a capillary volt of 13 V, sheath and auxiliary nitrogen gas velocities of 50 a.u. and 10 a.u. in total-scanning manner. TLC was carried out on precoated Si gel 60 F254 plates (Merck) and RP18 F254s plates (Merck). Spots were detected under UV (254 nm and 365 nm) before and after spraying with 10% H2SO4 ethanol solution, followed by heating the plate at 110° for 5 min. Column chromatography was carried out on wakogel C-100 (Wako Pure Chemical Industries Ltd.). The HPLC separation was performed on NPHPLC and RPHPLC; the former consisted of the following parts: a Merck-Hitachi system (L-7100 pumps, L-7400 UV detector, L-7490 RID, L-7300 column heater, L-7200 autosampler and D-7000 interface) and a Aquasil SS-4251 (60) column (10 mm×250 mm); the latter was composed by the following parts: a Shimadzu system (LC-6AD pump, SPD10Avp, UV detector, RID-10A detector, CTO-10ACvp column heater, SCL-10Avp system controller and SCL10Advp auto injector) and a Cosmosil 5C18-AR- Waters column (10 mm×250 mm, Japan Nacalai Tesque).

Plant material

The plant material was supplied by Jilin Hua Kang Pharm. Co. Ltd., China, in May 2001 and has been identified to be the herb of S. cannabifolius L. in July 2001 by the researcher Dr. Zhong-Gai Yan (Changchun Research Institute of Traditional Chinese Materia Medica, Jilin Province, China).

Extraction and isolation

The plant material was dipped into water overnight (1:10) and then extracted for three times after the material had been boiled for 2 h. Filtration and rotary evaporation of the resulting extract gave a residue (780 g), which was dissolved in water and extracted with chloroform, ethyl acetate, and n-butanol. The n-butanol layer was passed through a lowbar ODS column (1.873 g) eluted with MeOH-H2O (5:95, 1:9 v/v) and by HPLC with 15%MeOHH2O-1%THF to furnish 1 (31.5 mg, fig. 1), 2 (16.3 mg, fig. 2), 3 (11.6 mg, fig. 3), and 4 (11.5 mg, fig. 4).

Figure

Fig. 1: Compound 1.

Figure

Fig. 2: Compound 2.

Figure

Fig. 3: Compound 3.

Figure

Fig. 4: Compound 4.

Compound 1

Elution of the column with MeOH-H2O (5:95) afforded yellow liquid 1. IR υmax (KBr): 3396, 2928, 1710, 1669, 1365, 1242, 1157, 1077 cm-1. HRESIMS (positive) m/z: 403.1959[M+H]+ (calcd. for C19H31O9, 403.1963). 1H-NMR (400 MHz, DMSO-d6): δ5.76 (1H, s, H-1), 2.11 (3H, s, CH34), 2.25 (1H, dd, J=2.0; 12.8 Hz, H2-3’a), 1.26 (1H, m, H23’b), 4.16 (1H, m, H-4'), 1.97 (1H, dd, J=2.4; 12.4 Hz, H2-5'a), 1.35 (1H, m, H2-5'b), 1.28 (3H, s, CH3-7'), 1.31 (3H, s, CH38'), 1.07 (3H, s, CH3-9'), 5.09 (1H, s, OH-2'), 4.24 (1H, d, J=8.0 Hz, H-1'’), 2.89 (1H, dt, J=8.0; 4.4 Hz, H-2'’), 3.07 (1H, m, H-3'’), 3.13 (1H, m, H-4'’), 3.10 (1H, m, H-5'’), 3.66 (1H, ddd, J=2.0; 5.2; 10.4 Hz, H2-6'’a), 3.45 (1H, ddd, J=5.2; 5.2; 10.4 Hz, H2-6'’b), 4.88 (1H, d, J=4.4 Hz, OH-2'’), 4.88 (1H, m, OH-3'’), 4.91 (1H, m, OH-4'’), 4.46 (1H, t, J=5.2 Hz, OH-6'’). 13C-NMR (100 MHz, DMSO-d6): δ99.6 (C-1), 208.9 (C-2), 197.6 (C-3), 26.2 (C-4), 118.4 (C-1'), 70.4 (C-2'), 46.0 (C-3'), 70.6 (C-4'), 46.9 (C-5'), 35.5 (C-6'), 30.3 (C-7'), 28.7 (C-8'), 31.6 (C-9'), 101.2 (C-1'’), 73.4 (C-2'’), 76.7 (C-3'’), 70.0 (C-4'’), 76.7 (C-5'’), 61.0 (C-6'’).

Compound 2

Elution of the column with MeOH-H2O (1:9) afforded yellow liquid 2. IR υmax (KBr): 3423, 2927, 1668, 1374, 1076 cm-1. HRESIMS (positive mode) m/z: 387.2011[M+H]+ (calcd. for C19H31O8, 387.2013). 1H-NMR (400 MHz, DMSO-d6): δ2.00 (1H, dd, J=5.6; 12.8 Hz, H2-5a), 1.75 (1H, t, J=12.8 Hz, H2-5b), 4.25 (1H, m, H-6), 6.30 (1H, d, J=16.0 Hz, H-7), 5.80 (1H, dd, J=6.4; 16.0 Hz, H-8), 4.45 (1H, p, J=6.4 Hz, H-9), 1.32 (3H, d, J=6.4 Hz, CH3-9), 1.81 (3H, s, CH3-2), 1.13 (3H, s, CH3-4), 1.30 (3H, s, CH3-4), 5.12 (1H, br.d, J=3.6 Hz, OH-6), 4.29 (1H, d, J=7.6 Hz, H1'), 3.01 (1H, m, H-2'), 3.10 (1H, m, H-3'), 3.14 (1H, m, H4'), 3.20 (1H, m, H-5'), 3.66 (1H, ddd, J=1.6; 6.0; 12.0 Hz, H2-6’a), 3.51 (1H, ddd, J=6.0; 6.0; 12.0 Hz, H2-6'b), 5.06 (1H, d, J=4.4 Hz, OH-2'), 4.95 (1H, d, J=2.4 Hz, OH-3'), 5.00 (1H, br.s, OH-4'), 4.45 (1H, br.d, J=6.0 Hz, OH-6'). 13C-NMR (100 MHz, DMSO-d6): δ199.9 (C-1), 126.9 (C-2), 159.3 (C-3), 36.3 (C-4), 45.7 (C-5), 68.5 (C-6), 125.4 (C-7), 139.5 (C-8), 75.0 (C-9), 20.7 (C-10), 13.4 (C-11), 30.0 (C-12), 25.5 (C-13), 101.1 (C-1'), 73.7 (C-2'), 76.8 (C-3'), 69.7 (C-4'), 76.7 (C-5'), 60.8 (C-6').

Compound 3

Elution of the column with MeOH-H2O (1:9) afforded pale yellow oil 3. [α]20D+101.2° (c 0.12, MeOH). UV λ max (MeOH): 236 nm. IR υmax (KBr): 3420, 2924, 1653, 1373, 1040 cm-1. HRESIMS (positive) m/z: 387.2010[M+H]+ (calcd. for C19H31O8, 387.2013). 1H-NMR (400 MHz, DMSO-d6): δ2.04 (1H, d, J=16.4 Hz, H2-2a), 2.56 (1H, d, J=16.4 Hz, H2-2b), 5.75 (1H, s, H-4), 5.94 (1H, d, J=15.2 Hz, H-7), 5.63 (1H, dd, J=6.4; 15.2 Hz, H-8), 4.42 (1H, p, J=6.4 Hz, H-9), 1.18 (3H, d, J=6.4 Hz, CH3-9), 0.92 (3H, s, CH3-1), 0.91 (3H, s, CH3-1), 1.81 (3H, s, CH3-5), 4.99 (1H, s, OH-6), 4.08 (1H, d, J=7.6 Hz, H-1'), 2.95 (1H, m, H-2'), 2.93 (1H, m, H-3'), 3.02 (1H, m, H-4'), 3.04 (1H, m, H-5'), 3.63 (1H, p, J=6.0 Hz, H2-6’a), 3.40 (1H, m, H2-6’b), 5.11 (1H, d, J=4.4 Hz, OH-2'), 4.96 (1H, d, J=3.6 Hz, OH-3'), 4.86 (1H, d, J=4.4 Hz, OH-4'), 4.47 (1H, t, J=6.0 Hz, OH6'). 13C-NMR (100 MHz, DMSO-d6): δ40.9 (C-1), 49.3 (C2), 197.1 (C-3), 125.4 (C-4), 163.6 (C-5), 77.8 (C-6), 131.5 (C-7), 131.3 (C-8), 71.9 (C-9), 22.1 (C-10), 23.1 (C-11), 24.1 (C-12), 18.6 (C-13), 99.8 (C-1'), 73.2 (C-2'), 77.1 (C-3'), 70.0 (C-4'), 76.9 (C-5'), 61.0 (C-6').

Compound 4

Elution of the column with MeOH-H2O (1:9) afforded pale yellow oil 4. [α]20D-24.5° (c 0.11, MeOH). UV λmax (MeOH): 238 nm. IR υmax (KBr): 3418, 2927, 1654, 1373, 1075 cm-1. HRESIMS (positive) m/z: 387.2009[M+H]+ (calcd. for C19H31O8, 387.2013). 13C-NMR data (100 MHz, DMSO-d6): δ40.3 (C-1), 49.3 (C-2), 197.1 (C-3), 125.5 (C4), 163.8 (C-5), 77.7 (C-6), 130.2 (C-7), 133.1 (C-8), 74.5 (C9), 20.8 (C-10), 23.0 (C-11), 24.0 (C-12), 18.9 (C-13), 100.7 (C-1'), 73.6 (C-2'), 76.7 (C-3'), 69.9 (C-4'), 76.3 (C-5'), 61.0 (C-6').

Antibacterial bioassay

Three microbial cultures – Staphylococcus aureus IFO 3060, Bacillus subtilis and Escherichia coli – were supplied by Faculty of Agriculture, Kochi University, microbial laboratory culture collection. Got proper amount of each fraction to form the solution of the crude drug (100 g/ml) with 80% ethanol and diluted it with sterile distilled water before the experiment. Transferred 100 μl of sample solution into the test tube 1 and diluted it with 900 μl of distilled water in order to change the concentration to be 10 g/ml. Drew another 100 μl of solution from tube 1, then repeated the procedure as mentioned before to obtain the sample solution in tube 2 with concentration of 1 g/ml.

The qualitative antibacterial bioassay of each extract employed was the agar diffusion assay. LB liquid medium was prepared by mixing 10 g of peptone, 5 g of yeast, and 5 g of NaCl solution together in 1000 ml distilled water, adjusting pH value to be 7.2 by 2 N NaOH solution. Then added 15 g of agar to LB liquid medium to obtain LB agar solid medium. The bacteria were incubated in three test tubes with 3 ml of LB liquid medium in under shaking at 37° for 18 h. The inocula of target strains were injected into the LB agar solid medium sterilized at 121° for 15 min (100 μl of each inoculum). Then dispensed the agar medium into a sterilized culture dish with seven stainless steel wells (interior diameter: 6 mm, external diameter: 8 mm and height: 10 mm). After the medium was solidified, got rid of the wells, added 100 μl of sample solution to each well, and repeated with every diluted concentration (10 g/ml and 1 g/ml). Finally, sealed the culture dish. The diameters of the inhibition zones were measured using a hand-held digital calliper after incubation for 24 h at 30°. The mean values count (two times).

MIC values of purified compounds were determined by the turbidimetry. Just the right amount of the purified compound was dissolved in DMSO to achieve the solution with concentration of 10 mg/ml. Drew 500 μl of solution by a micropipette before transferring to a graduated centrifuge tube, diluted the solution (1:1) with sterile distilled water, and mixed under shaking by the mini-oscillator to change the concentration to be 5 mg/ml. Then drew another 500 μl of solution and transferred to next centrifuge tube before diluting it with 500 μl of distilled water. Repeated the operation as mentioned above. As a result, a series of the sample solution in decreasing concentration was obtained by a ratio of 0.5 (final concentration: 10 mg/ml to 9.78 μg/ml). Drew 100 μl of solution from each sample of different concentration and added the solutions into the test tubes that have already been prepared by pouring 900 μl of LB liquid medium into them. After the inocula of target strains were uniformly added (20 μl), put on the lid of tubes and incubated them at 30° under shaking for 48 h. Growth was followed by subjecting to UV spectrophotometry (Shimadzu UV-1200) at 600 nm using DMSO as control to measure the absorbance. The MIC value was defined as the lowest concentration to inhibit visible growth. DMSO, at used concentration in the test, did not interfere with the microbial growth.

Results and Discussion

Compound 1 was isolated as a yellow liquid. The Molish’s reaction was positive, indicating that this compound was glycoside. The molecular formula of 1 was established as C19H30O9 on the basis of the protonated molecular ion peak [M+H]+ at m/z 403.1959 and the NMR data, allowing five degrees of unsaturation. The 13C-NMR spectrum showed 18 carbon signals, of which 6 carbon signals were assigned to a sugar portion, but the O-bearing carbon signal at δ76.7 (C-3'’ and C-5'’) was extremely strong, implying the degeneration of the two carbon signals, which was further supported by the correlations from three protons at δ3.01-3.15 (3H, m) to two methine carbons (δ70.0 and 76.7). Considering the result of subjecting the hydrolyzate of 1 and authentic sample of glucose to PPC and the anomeric proton resonance at δ 4.24 (1H, d, J=8.0 Hz, H-1'’), the sugar was identified as β-D-glucose. Correlation was observed from the lone proton at δ5.76 (1H, s) to δ28.7 (C-8'), 35.5 (C-6'), 70.4 (C2'), 118.4 (C-1'), 197.6 (C-3) and 208.9 (C-2). Two three- proton singlets of four methyl groups at δ1.07 (3H, s) and 1.31 (3H, s) were meta-methyl moieties attached to a quaternary carbon (δ c 35.5). The HMBC of 1 showed connectivities from H-7' to C-3', C-2' and C-1'. In the 1H NMR spectra, five exchangeable proton resonances at δ 5.09 (1H, s), 4.91(1H, m), 4.88 (2H, m), 4.46 (1H, t, J=5.2 Hz) were assigned to OH-2', OH-4'’, OH-3'’, OH-2'’ and OH-6'’.

The HMBC connectivities from the anomeric proton at δ 4.24 to C-4' (δ70.6) allowed the glucose to attach at C-4'. The novel compound was thus assigned the structure 1(2-hydroxy- 2, 6, 6-trimethyl-4-β-D-glucosyloxy-cyclohexylidene)-butane-2,3-dione, which was reported for the first time and named cannabiside D.

Compound 2, obtained as a pale yellow oil, was found to have a molecular formula of C19H30O8 as determined by HRESIM, implying five elements of unsaturation. The Molish’s reaction was positive, indicating that this compound was glycoside. From the coupling constant of anomeric proton at δ4.29 (1H, d, J=7.6 Hz) and the hydrolysate, compound 2 was identified as β-Dglucopyranoside. 1H-NMR contained five exchangeable proton resonances at δ5.12 (1H, br.d, J=3.6 Hz), 5.06 (1H, br.d, J=4.4 Hz), 5.00 (1H, br.s), 4.95 (1H, d, J=2.4 Hz) and 4.45 (1H, d, J=6.0 Hz), characteristic of hydroxyl functions, four of which must locate at the glucose, and the proton (δ5.12) should bond to C-6 (δ68.5) because of long-range connectivities observed from the hydroxyl proton at δ5.12 to carbonyl carbon (δ199.9), methine carbon (δ68.5), and methylene carbon (δ45.7). There was also discerned correlation from methyl protons at δ1.81 (3H, s) to carbonyl carbon (δ199.9) and sp2 hybridized quaternary carbon (δ159.3 and 126.9). A series of proton signals at δ6.30 (1H, d, J=16.0 Hz) and 5.80 (1H, dd, J=16.0; 6.4 Hz) observed in alkenyl proton region of the 1H-NMR spectrum were affirmed to be trans-alkenyl protons coupling with each other and assigned to methine protons at δ125.4 and 139.5 by the DEPT 135° spectrum. Carbons (δ139.5 and 125.4) were correlated with methine proton at δ4.45 (1H, p, J=6.4 Hz) and long-range connectivities were observed from methyl protons at δ1.32 (3H, d, J=6.4 Hz) to C-8 (δ139.5) and C-9 (δ75.0).In NMR, spectra indicated two lone methyl groups at δ30.0 [1.13 (3H, s)] and 25.5 [1.30 (3H, s)], between which, marked correlation was displayed in HMBC. Strong long-range connectivites were observed from methyl protons at δ1.13 (3H, s) and 1.30 (3H, s) to C-3 (δ159.3), C-4 (δ36.3), and C-5 (δ45.7); from alkenyl proton at δ6.30 (1H, d, J=16.0 Hz) to C-2 (δ126.9); and from alkenyl proton at δ5.80 (1H, dd, J=6.4; 16.0Hz) to C-3 (δ159.3).

The anomeric proton at δ4.29 (1H, d, J=7.6 Hz) was long-range connected to C-9 (δ75.0) in HMBC, suggesting the glycoside was formed between the glucose and OH-9. Thus by the analysis of above evidence, compound 2 was determined to be 6-Hydroxy-3-(3-O-β-Dglucopyranosyl- but-enyl)-2,4,4-trimethyl-cyclohex-2-enone, which has not been reported before and named cannabiside E.

Compound 3 was yielded as a pale yellow oil and analyzed for C19H30O8 by HREMIS. The positive Molish’s reaction and acid hydrolysis of 3 indicated it to be β-D-glucopyranoside in accordance with the coupling constant of anomeric proton at δ4.08 (1H, d, J=7.6 Hz). Of the five exchangeable protons at δ5.11 (1H, d, J=4.4 Hz), 4.96 (1H, d, J=3.6 Hz), 4.86 (1H, d, J=4.4 Hz), 4.47 (1H, t, J=6.0 Hz) and 4.99 (1H, s), the last one was a lone active proton linked with a quaternary carbon, and the other protons were resided at the glucose. The alkenyl proton region in 1H NMR spectrum contained a series of proton signals at δ5.94 (1H, d, J=15.2 Hz) and 5.63 (1H, dd, J=15.2; 6.4Hz) whose chemical shifts and large coupling constants implied them to be coupling trans-alkenyl protons in the DEPT 135° spectrum; therefore, the isolated natural product was confirmed to be 9-O-β-Dglucopyranosyloxy-6-hydroxy-3-oxo-α-ionol. Consulting the NMR data of the known compound (6S, 9S)roseoside [4,5], the compound was identified as (6S, 9S)roseoside, which was isolated for the first time in genus Senecio.

Compound 4 was obtained as a pale yellow oil with the positive Molish’s reaction, indicating it to be glycoside. Comparing its 13C NMR spectrum with that of compound 3, the two spectra are almost the same, except for the methine carbon (δ74.5). As a result, compound 3 and 4 were determined to be epimers. The identification of their absolute stereo-configuration is under consideration.

By the antibacterial (screening) bioassay in vitro, the CHCl3, EtOAc and n-BuOH soluble fractions, except for the water layer, showed the potential antibacterial activities. The minimum inhibitory concentrations (MICs) of all tested substances toward several pathogenic bacteria are shown in Table 1.

Comp. Minimum inhibition concentration (MIC)
Staphylococcus Bacillus aureus Escherichia subtiliscoli
1 3.9 15.6 >1000
2 125.0 250.0 >1000
3 31.2 62.5 >1000
4 31.2 31.2 >1000

Table 1: Antibacterial activity of compounds 1-4.

Compounds 1-4 were all active at concentrations less than 250.0 μg/ml against two gram-positive organisms (Staphylococcus aureus IFO 3060, Bacillus subtilis), but none were active against the gram-negative organism (Escherichia coli), being MIC values for the strain greater than 1000 μg/ml by employing turbidimetry.

Acknowledgements

The authors are grateful to Master Katsuyosi Tanaka, Faculty of Agriculture, Kochi University, for providing technical support to this study. The work was supported by “Shiwu Grand Technological Special Item” of China (No. 2003AA2Z3257).

References