Three antibacterial compounds from the roots of Pteris multifida

Article abstract of DOI:10.1007/s10600-009-9258-5

A new eudesmane-type sesquiterpenoid, 3β-caffeoxyl-1β,8α- dihydroxyeudesm-4(15)-ene (1), together with two known compounds including ludongnin V (2) and isoneorautenol (3), were isolated from the roots of Pteris multifida. Their structures were determined

Full text of DOI:10.1007/s10600-009-9258-5

Chemistry of Natural Compounds, Vol. 45, No. 1, 2009
THREE ANTIBACTERIAL COMPOUNDS
FROM THE ROOTS OF Pteris multifida
Hu Hao-bin,1* Zheng Xu-dong,
1
UDC 547.596.7
1
2
Hu Huai-sheng, and Cao Hong
A new eudesmane-type sesquiterpenoid, 3β-caffeoxyl-1β,8α-dihydroxyeudesm-4(15)-ene (1), together with
two known compounds including ludongnin V (2)and isoneorautenol (3), were isolatedfromthe roots of Pteris
multifida. Their structures were determined by spectral and chemical methods, with their antibacterial
activities being evaluated by the microdilution technique, respectively.
Key words: Pteris multifida, Pteridaceae, eudesmane sesquiterpenoid, antibacterial activity.
Pteris multifida (Pteridaceae) is a perennial herb growing mainly in the south and southwest regions of China [1]. It
is used in traditional Chinese medicine as an antimicrobial agent for the treatment of eczema, hematemesis, rheumatism,
enterits, diarrhea, cold, and so on [2, 3], and its extracts also show high antimutagenic activityagainst picrolonic acid-induced
mutation [4]. Previous chemical examination of the roots of P. multifida afforded saucerneol D, β-rosaterol,
dehydrogoniothalamin, muxiangrine III, etc. [5–7]. In order to investigate the chemical composition and the antibacterial
potential of P. multifida in more depth and define the chemodiversity of the plant and the consequent potential value of this
natural resource, as well as to establish the best procedure to obtain extracts containing active principles, we have again isolated
three active compounds, including a new eudesmane-type sesquiterpenoid and two known spirosecokaurene diterpenoid and
furocoumarin from P. multifida. In this paper, we wish to report the isolation and structure elucidation as well as the
antibacterial properties of these three compounds.
Compound 1 was obtained as a pale yellow amorphous powder from MeOH. The molecular formula was established
+
as C H O from the HREI-MS data at m/z 416.2204 [M] (calcd for 416.2199), corresponding tonine degrees ofunsaturation,
2
4 32 6
ascribed to one carbonyl group, one benzene ring, two double bonds, and two alicyclic rings with the aid of spectral data. It
exhibited UV maxima at 216, 220, 254, 298, and 325 nm, suggesting the presence of strong conjugation in the molecule. A
bathochromic shift of 47 nm upon addition of NaOH indicated the presence of free phenolic hydroxy. A distinct bathochromic
shift with AlCl but again returning to the normal value upon addition of HCl indicated the presence of a catechol group. The
3
–
1
IR spectrum exhibited absorption bands for hydroxyl groups (3408–3100 cm , br), α,β-unsaturated ester moiety (1695 and
270 cm ), isopropyl group (1358, 1160 cm ), exocyclic double bond (1650, 1475 cm ), and aromatic rings (1600 and
510 cm ). Analysis of the NMR spectra with the aid of the DEPT technique demonstrated the presence of a caffeoyl group
–
1
–1
–1
1
1
–
1
[
including a trans-conjugated double bond signal at δ 6.38 (1H, d, J = 16.0 Hz) and 7.64 (1H, d, J = 16.0 Hz), which correlated
H
with signals at δ 115.8 and 147.0, an ester carbonyl (δ 170.8), and a typical ABX aromatic ring spin system signal at δ 6.77
C
C
H
(
1H, d, J = 8.1 Hz), 6.95 (1H, dd, J = 8.1, 2.0 Hz) and 7.04 (1H, d, J = 2.0 Hz), corresponding to δ 127.8, 115.1, 145.9, 149.2,
C
1
16.1 and 122.8] (Table 1). Besides the nine carbon signals for the caffeoyl group, 15 additional carbon and corresponding
proton signals, including three methyls [one tertiary methyl (δ 12.3 and δ 0.72) and two secondary methyl groups (δ 14.8
C
H
C
and 20.6; δ 1.03 and 0.91)], three alicyclic methylenes [δ 31.8, 25.9, 37.2; δ 1.80, 1.90, 1.67], one exocyclic olefinic
H
C
H
terminal methylene [δ 107.7; δ 4.82 and 4.96], one exocyclic olefinic quaternary carbon [δ 148.6], six methines [two
C
H
C
alicyclic methines (δ 56.5, 39.4; δ 1.75, 2.35), one isopropyl methine (δ 31.4 and δ 1.71), three oxygenated methines [δ
C
H
C
H
C
1
13
7
8.0, 81.6, 80.7; δ 3.66, 5.12, 3.24], and one alicyclic quaternary carbon [δ 49.6] were recognized in the H and C NMR
H
C
(
DEPT) spectra.
1
) College of Chemistry and Chemical Engineering, Longdong University, Qingyang 745000, P. R. China, fax: +86
9
34 8632822, e-mail: hhb-88@126.com, huhaobin_88@yahoo.com.cn; 2) Department of Life Science, Longdong University,
Qingyang 745000, P. R. China. Published in Khimiya Prirodnykh Soedinenii, No. 1, pp. 41-43, January-February, 2009.
Original article submitted August 15, 2007.
©
0
009-3130/09/4501-0045 2009 Springer Science+Business Media, Inc.
45

1
13
TABLE 1. H and C NMR Chemical Shifts of Compound 1 in CDCl , SiMe as Internal Standard (δ, ppm, J/Hz)
3
4
C atom
δC
δH
C atom
δC
δH
0.91 (3H, d, J = 6.9)
0.72 (3H, s)
4.82 (1H, d, J = 6.9)/4.96 (1H, d, J = 6.9)
1
2
3
4
5
6
7
8
9
78.0
31.8
81.6
148.6
56.5
25.9
39.4
80.7
37.2
49.6
31.4
14.8
3.66 (1H, dd, J = 11.0, 4.5)
1.80 (1H, m)/1.51 (1H, m)
5.12 (1H, dd, J = 6.0, 10.0)
-
1.75 (1H, d, J = 10.1)
1.90 (1H, m)/1.32 (1H, m)
2.35 (1H, m)
3.24 (1H, d, J = 9.8)
1.67 (1H, m)/1.38 (1H, m)
-
13
14
15
1′
2′
3′
4′
5′
6′
7′
8′
9′
20.6
12.3
107.7
127.8
115.1
145.9
149.2
116.1
122.8
147.0
115.8
170.8
-
7.04 (1H, d, J = 2.0)
-
-
6.77 (1H, d, J = 8.1)
6.95 (1H, dd, J = 8.1, 2.0)
7.64 (1H, d, J = 16.0)
6.38 (1H, d, J = 16.0)
-
1
1
1
0
1
2
1.71 (1H, m)
1.03 (3H, d, J = 6.9)
TABLE 2. MIC Values of Compounds 1-3 in μg/mL
Gram-positive bacteria
Compound
Gram-negative bacteria
B. sphaeriicus
B. subtilis
S. aureus
E. coli
P. aeruginosa
S. typhimurium
1
2
3
75
75
75
50
25
25
25
100
12.5
50
100
50
50
25
100
100
12.5
100
100
6.25
No activity
12.5
Ampicillin
6.25
3.13
OH
1
H
CH
H
H
1
4
3
OH
H
8
H
H
HO
HO
OH
H
H
H
O
7'
3
OH
CH
3
9'
7
12
H
HO 3'
HO
1'
O
O
4
1
1
3
H
1
5H
H
CH
1
3
O
H
Fig. 1. Key HMBC and NOE correlations of compound 1.
These spectral data suggested that compound 1 has a bicyclic sesquiterpene moiety possessing two hydroxyl groups
and an exocyclic double bond in an eudesmane-type framework. The NMR spectra of the sesquiterpene moiety showed a very
closesimilaritytothose ofeudesm-4 (15)-ene-1β,8α-diol [8], the onlydifference being an additional caffeoxyl groupatC-3from
the HMBC correlations between H-3 with C-9′/C-2/C-4/C-15. This conclusion was further supported bythe observed downfield
shifts of C-3 (δ 81.6) and H-3 (δ 5.12) with respect to the corresponding signals in eudesm-4 (15)-ene-1β,8α-diol [8] and
C
H
eudesm-4(15)-ene-1β,6α-diol [9]. The β-configuration of the 3-caffeoxyl group was determined from the chemical shifts and
coupling constants of H-3 (5.12, dd, J = 6.0, 10.0 Hz), and further evidenced by the observed NOE interactions between H-3
and H-1/H-5 in the ROESY spectrum (Fig. 1). In turn, the HMBC correlations between H-1 and C-5/C-3, H-8 and C-6/C-7/C-
9
/C-10/C-11, H-6 and C-5/C-7/C-10/C-11, H-12 and C-7/C-13, H-15 and C-3/C-5, as well as H-14 and C-1/C-5/C-9, clearly
indicated the attachment ofthe hydroxyl, isopropyl, exocyclic ethylenic bond, and angular methyl functional groups at C-1, C-8,
C-7, C-4, and C-10, respectively. The observed NOE interactions between H-5 and H-1/H-3/H-7/H -9, H -14 and H -2/H -
ax
3
ax
ax
6
/H-8/H -9, H-8 and H -6/H -12, and H-7 and H-5/H -9/H -13 distinctlyindicated that the configurations of HO-1, CH -10,
ex ax 3 ax 3 3
and C-7 isopropyl must be β-oriented. H-5 and HO-8 were α-oriented, suggesting that the A/B ring juncture was trans [10]. The
4
6

correlations between H-2′ and C-7′, H-7′ and C-6′/C-9′, H-8′ and C-1′/C-9′, H-2′ and H-7′, as well as H-6′ and H-8′, also were
clearly observed in the HMBC and ROESY experiments.
On alkaline hydrolysis, compound 1 also yielded eudesm-4 (15)-ene-1β,8α-diol [8] and caffeic acid, which was
compared with an authentic sample by melting point and co-TLC. Therefore, based on the above spectral features and
physicochemical properties, thestructureofcompound1wascharacterizedas3β-caffeoxyl-1β,8α-dihydroxyeudesm-4(15)-ene,
which has not been reported previously from any plant source.
Compound 1 showed moderate activity against both gram-positive and gram-negative bacteria (Table 2).
Compound 2 was active against S. typhimurium, E. coli, and S. aureus, but less so against the remaining organisms.
Compound 3 had a minimum inhibitory concentration (MIC) of less than 50 μg/mL against S. aureus and
P. aeruginosa, while it had no activity against S. typhimurium. In comparison with standard antibiotics, the activities of the
compounds were not as promising, but the presence of these active constituents may contribute to the antibacterial and
antiinflammation effects.
EXPERIMENTAL
General Experimemtal Procedures. Melting points were measured on a Chinese X-4 melting point apparatus
(
uncorrected), UV spectra were recorded on a Shimadzu UV-240 spectrometer, IR spectra (KBr disks) were obtained on an
Alpha-Centari FT-IR spectrometer, NMR spectra were scanned on a Bruker AM-500 spectrometer (chemical shifts in δ
1
13
downfield from TMS internal standard) operating at 500 and 125 MHz for H and C NMR respectively, and EI-MS spectra
on JMS-02SB mass spectrometer.
Plant Material. P. multifida roots were collected at the flowering stage, in July to August 2002, from the Pingjiang
District ofHunan Province(China), and authenticated byProf. Yun-Shan Lian in the Department ofBiology, Northwest Normal
University. A voucher specimen (No.107083) of the plant was deposited in the Herbarium of the Faculty of Biology, Northwest
Normal University, Lanzhou 730070, China.
Extraction and Isolation. The air-dried roots of P. multifida (1 kg) were ground and extracted with MeOH. The
concentrated extract was partitioned with petroleum ether, EtOAc, and n-BuOH. The EtOAc-soluble part was chromatographed
over silica gel with the CHCl –Me CO (25:1→1:20, V/V) gradient system repeatedly, recrystallized, and purified on Sephadex
3
2
LH-20 with CHCl -MeOH (5:1) to get 1 (8 mg), 2 (16 mg), and 3 (13 mg).
3
3
β-Caffeoxyl-1β,8α-dihydroxyeudesm-4(15)-ene (1): C H O , mp 204–206°C (MeOH). UV(λ , nm), +MeOH:
24 32 6 max
–
1
2
3
1
16, 220, 254, 298, 325; +NaOH: 256, 372; +AlCl : 248, 304sh, 387; +AlCl +HCl: 228, 262sh, 296, 324. IR (KBr, ν, cm ):
408-3100, 1695, 1650, 1600, 1510, 1475, 1358, 1270, 1160, 820; EI-MS: m/z 416 [M] (5.2), 236 (1.8), 218 (12.4), 175 (8.9),
63 (100), 109 (23.1); H and C NMR are listed in Table 1.
3 3
+
1
13
+
Ludongnin V (2): colorless flaky crystals (CHCl –Me CO), mp 258–260°C, HREI-MS m/z 360.1567 [M] (calcd for
3
2
3
60.1573, C H O ). Comparison of the spectral (IR and NMR) data with the literature identified it as ludongnin V [11].
20 24 6
+
Isoneorautenol (3): white plate crystals (Me CO), mp 153–155°C, HREI-MS m/z 322.1211 [M] (calcd for 322.1205,
2
C H O ). The physicochemical constants and spectral (IR, NMR, and MS) data of 3 indicate that it is identical to
2
0 4
18
isoneorautenol [12].
Alkaline Hydrolysis of Compound 1. Compound 1 (5 mg) was refluxed with 5% KOH–MeOH solution (5mL) under
heating at 90°C for 3 h. After cooling, the reaction mixture was diluted with H O (10 mL), then extracted with CHCl . The
2
3
organic layers was dried with Na SO and evaporated to afford colorless needle crystals of eudesm-4 (15)-ene-1β,8α-diol: mp
2
4
1
06–108° (lit. 105–106°C), which exhibited comparable NMR spectral data to literature values [8]. The aqueous layer was
neutralized with 5% HCl and then extracted with Et O (twice); the ether layer was concentrated under pressure and
2
recrystallized to give light-yellow plate crystals of caffeic acid, which was identified by comparison of the mixed melting point
and R value [R 0.43, byco-TLC using CHCl –MeOH–HCOOH (9:1: 0.5, V/V) as developing solvent] with authentic samples.
f
f
3
Antimicrobial Screening. Antimicrobial screening of the compounds was performed by using the microdilution titer
technique [13] to determine MICs of the metabolites against three gram-positive bacteria: Bacillus sphaericus (ATCC 14577),
Bacillus subtilis (ATCC 6051), and Staphylococcus aureus (ATCC 6538), and three gram-negative bacteria: Escherichia coli
(ATCC 10538), Pseudomonas aeruginosa (ATCC 25619), and Salmonella typhimurium (ATCC 23564). In the test, ampicillin
is used as a positive control and resazurin as a color indicator.
47

ACKNOWLEDGMENT
The authors are grateful to Prof. Zheng Shang-zhen for experimental assistance, and Prof. Lian Yong-shan for
identifing the plant sample. This research was supported bythe Science Research Project ofLongdong University(XYZK0503).
REFERENCES
1
.
.
Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita, Flora Reipublicae Popularis
Sinicae, V. 3, Science Press, Beijing, 1990, 40.
Jiangsu New Medical College, Dictionary of Traditional Chinese Drugs, Science and Technology Press, Shanghai,
2
1
985, 487.
3
4
5
6
7
8
9
.
.
.
.
.
.
.
H. R. Zhang, W. S. Wu, H. S. Lu, and M. Z. Wang, Subtrop. Plant Res. Commun., 29, 5 (2000).
H. Lee and J.-Y. Lin, Mutat. Res./Genet. Toxicol., 204, 229 (1998).
H. B. Hu and X. D. Zheng, Acta Bot. Boreal.-Occident. Sin., 25, 781 (2005).
H. B. Hu and X. D. Zheng, Nat. Prod. Res. Dev., 16, 379 (2004).
H. B. Hu and X. D. Zheng, Nat. Prod. Res. Dev., 17, 169 (2005).
Z. H. Sun, S. Zhang, J. Shi, and C. Q. Hu, Chin. J. Org. Chem., 24, 7 (2004).
F. Bohlmann, N. Ates, R. M. King, and H. Robinson, Phytochemistry, 22, 7 (1988).
A. A. Ahmed, A. M. Ahmed, T. A. Eptehal, T. Olga, C. Maria, J. M. Tom, G. Tamas, and T. Gabor,
Phytochemisry, 59, 851 (2002).
1
1
0.
1.
Y. B. Yang, S. Q. Liu, B. D. Wang, C. Zhu, D. Y. Zhu, and X. M. Kong, Chin. J. Appl. Environ. Biol., 3, 79
(
1997).
1
1
2.
3.
L. A. Mitscher, S. K. Okwute, S. R. Gollapudi, S. Drake, and E. Avona, Phytochemistry, 27, 3449 (1988).
M. M. Rahman and A. I. Gray, Phytochemistry, 59, 73 (2002).
4
8