New antimycobacterial saponin from Colubrina retusa

Article abstract of DOI:10.1021/np9901940

A new jujubogenin saponin was isolated from the stems of Colubrina retusa and identified as jujubogenin 3-O-α-L-arabinofuranosyl (1→2)-[3-O- (trans)-p-coumaroyl-β-D-glucopyranosyl (1→3)]-α-L-arabinopyranoside (4) on the basis of chemical and spectroscopic data. The antimycobacterial activity expressed as minimum inhibitory concentration (MIC) for compound 4 was 10 μg/mL.

Full text of DOI:10.1021/np9901940

J . Nat. Prod. 1999, 62, 1341-1342
1341
New An tim ycoba cter ia l Sa p on in fr om Colu br in a r etu sa
H. N. ElSohly,*,† S. Danner,†,§ X.-C. Li,† A. C. Nimrod,† and A. M. Clark†,‡
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and Department of
Pharmacognosy, School of Pharmacy, Thad Cochran Research Center, The University of Mississippi,
University, Mississippi 38677
Received April 26, 1999
A new jujubogenin saponin was isolated from the stems of Colubrina retusa and identified as jujubogenin
3-O-R-L-arabinofuranosyl (1f2)-[3-O-(trans)-p-coumaroyl-â-D-glucopyranosyl (1f3)]-R-L-arabinopyranoside
(4) on the basis of chemical and spectroscopic data. The antimycobacterial activity expressed as minimum
inhibitory concentration (MIC) for compound 4 was 10 µg/mL.
Ta ble 1. 13C and 1H NMR Data for Compound 4 in Pyridine-d5
(ppm)
Colubrina retusa (Ditter) Cowan is a dicotyledonaceous
plant of the Rhamnaceae family, whose 55 genera and 900
species are widely distributed especially in the tropics. The
only rhamnaceous plants of some economic importance are
Ziziphus species and Rhamnus purshiana.1 A previous
study carried out on the stem of C. retusa2 resulted in the
isolation of a known saponin identified as jujubogenin [3-O-
R-L-arabinofuranosyl (1f2)-â-D-glucopyranosyl (1f3)]-R-
L-arabinopyranoside (1),3 along with two new saponins:
jujubogenin 3-O-R-L-arabinofuranosyl (1f2)-[2-O-(trans,
cis)-p-coumaroyl-â-D-glucopyranosyl (1f3)]-R-L-arabinopy-
ranoside (2) and jujubogenin 3-O-(5-malonyl)-R-L-arabino-
furanosyl (1f2)-[â-D-glucopyranosyl (1f3)]-R-L-arabinopy-
ranoside (3). To isolate new constituents from this plant,
further investigation of a column fraction yielded a new
saponin (4) whose structure was established by spectro-
scopic means. ESI-MS showed a molecular ion peak at m/z
1067 [M + Na]+ suggesting the molecular formula C55H80O19.
δC
δH (J , Hz)
δC
δH (J , Hz)
aglycone-1 39.2 1.54, 0.75
Ara (p)-1 105.4 4.75 (d, 7)
2
3
4
5
6
7
8
9
26.8 2.10, 1.79
88.9 3.21 (dd, 11.7, 4.3)
2
3
4
5
76.9 4.45
83.6 4.16
68.3 4.50
65.4 4.15, 3.65
40.0
56.7 0.66
-
18.4 1.45, 1.33
36.4 1.47
Glc(p)-1 104.8 5.14 (d, 7.8)
2
3
4
5
6
73.4 4.02
79.3 5.92
70.0 4.32
78.5 3.94
37.8
53.6 0.86
-
10 37.6
11 22.0 1.51
-
62.6 4.43, 4.22
12 28.7 1.91, 1.88
13 37.5 2.79
Ara (f)-1 110.4 6.03 (d, 2.8)
2
3
4
5
83.4 4.97
78.3 4.78
85.4 4.71
62.6 4.30, 4.14
14 53.9
15 37.0 2.42/1.49 (ABq, 8)
16 110.6
-
-
17 54.6 1.35
18 19.0 1.04 (s)
19 16.4 0.68 (s)
p-Coum-1 126.5 -
2,6 130.5 7.43 (d, 8.6)
3,5 116.9 7.10 (d, 8.6)
20 68.7
-
21 30.1 1.33 (s)
22 45.6 1.74
23 68.8 5.12
24 127.2 5.48
4 161.3 -
R- 115.9 6.49 (d, 15.9)
â- 145.1 7.85 (d, 15.8)
OdC 167.6
-
25 134.2
-
26 25.5 1.66 (s)
27 18.6 1.71 (s)
28 28.2 1.24 (s)
29 16.7 1.02 (s)
30 65.9 4.23
20-OH
5.93 (s)
presence of p-coumaroyl group was indicated by the carbon
signals at δ 115.9 (d), 116.9 (d), 126.5 (s), 130.5 (d), 145.1
1
(d), 161.3 (s), and 167.5 (s). The H NMR spectrum showed
the aromatic signals as two ortho-coupled doublets at δ 7.43
(2H, J ) 8.6 Hz) and 7.10 (2H, J ) 8.6 Hz) together with
another two doublets at δ 6.49 (1H, J ) 15.9 Hz) and 7.85
(1H, J ) 15.9 Hz) integrating for one proton each; these
were assigned to the olefinic protons, and the size of the
coupling constant indicated a trans configuration. Complete
assignments of the 1H and 13C NMR signals were ac-
complished by 2D NMR including COSY, HMQC, and
HMBC (Table 1). The linkage position of the p-coumaroyl
residue to the sugar moiety was determined by 2D NMR.
1H-1H COSY and HMQC established each sugar’s spin-
coupling network. The signal of H-3 of glucose in 4 was
significantly shifted to a downfield value of δ 5.92 when
compared with 1, in which it resonated at δ 4.22.2 This
indicated that the p-coumaroyl group was linked at the 3
position of glucose. Key HMBC correlations are sum-
1
Comparison of the H and 13C NMR spectra of 4 with the
previously identified saponins 1 and 2 suggested the same
type of jujubogenin aglycone. The 13C NMR of 4 displayed
three anomeric carbon signals at δ105.4, 104.8, and 110.4
as well as a typical carboxylic ester carbon signal at δ167.5
along with some aromatic and olefinic carbon signals.
Aromatic and olefinic proton signals were also observed in
the downfield region of the 1H NMR spectrum. On alkaline
hydrolysis, compound 4 afforded 1 and p-coumaric acid. The
* To whom correspondence should be addressed. Tel.: (601) 232-7610.
Fax: (601) 232-1006. E-mail: helsohly@olemiss.edu.
† National Center for Natural Products Research.
‡ Department of Pharmacognosy.
§ Current address: University of Vienna, Vienna, Austria.
10.1021/np9901940 CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/24/1999

1342 J ournal of Natural Products, 1999, Vol. 62, No. 9
Notes
of 17 combined fractions were obtained. The new saponin (4)
was obtained from fraction #5 (109 mg) by further purification
on HPLC using an isocratic mixture of 37% acetonitrile in
water at a flow rate of 3.5 mL/min (tR 19 min). This material
was dissolved in 1.5 mL of MeOH, and 24 injections of 40-65
µL each were made. The combined fractions were evaporated
to dryness to yield compound 4.
J u ju b ogen in 3-O-r-L-a r a b in ofu r a n osyl (1f2)-[3-O-
(tr a n s)-p -cou m a r oyl-â-D-glu cop yr a n osyl(1f3)]-r-L-a r a -
bin op yr a n osid e (4). White powder (26.5 mg), mp 212 °C,
1
[R]25 -79.7° (c, 0.025, MeOH); H and 13C NMR spectra are
D
listed in Table 1; ESI-MS m/z, 1067.5199 [M + Na]+ (calcd for
C
55H80019Na, 1067.5186).
F igu r e 1. Key HMBC correlations of 4.
Alk a lin e Hyd r olysis. A solution of 4 (1 mg) in 1% KOH
(0.2 mL) was kept at room temperature for 30 min. The
reaction mixture was subjected to TLC analysis using CHCl3/
MeOH/H2O (30:10:1) as a developing system and 10% H2SO4
as a chromogenic reagent. Saponin 1 was detected with an Rf
value of 0.72. For detection of the acid group, the reaction
mixture was acidified with 2 N HCl, evaporated to dryness
under a stream of N2 and the residue dissolved in a drop of
MeOH. The methanolic solution was spotted on TLC along
with reference p-coumaric acid and the plate developed in
CHCl3/MeOH (6:1). p-Coumaric acid with an Rf value of 0.47
was detected by UV.
marized in Figure 1. On the basis of the evidence above,
compound 4 was established to be jujubogenin 3-O-R-L-
arabinofuranosyl (1f2)-[3-O-(trans)-p-coumaroyl-â-D-glu-
copyranosyl (1f3)]-R-L-arabinopyranoside. Compounds 2
and 4 were active against Mycobacterium intracellulare
(MIC of 50 and 10 µg/mL, respectively); while 1 was
inactive.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
An tim ycoba cter ia l Assa y. Mycobacterium intracellulare
(ATCC#23068) was maintained on Lowenstein-J enson slants
at 4 °C. OADC-supplemented Middlebrook broth (5 mL) was
inoculated with bacteria and incubated at 37 °C for 72 h.
Compounds 1, 2, and 4 were solubilized in DMSO, further
diluted in sterile saline and a 1:30 diluted inoculum of the
bacteria in Middlebrook broth was added to the diluted
samples in the wells of round-bottomed microtiter plates. The
plates were incubated at 37 °C for 48 h and then visually
assessed for growth. The antimycobacterial activity expressed
as minimum inhibitory concentration (MIC) for compound 4
was 10 µg/mL. Rifampicin was used as a positive control (MIC
) 0.3 µg/mL).
determined on
a Thomas-Hoover capillary melting point
apparatus and are uncorrected. NMR were recorded in pyri-
dine-d5 with TMS as internal standard, using Bru¨ker Avance
DPX-300 (300 MHz) DRX-400 (400 MHz), and DRX-500 (500
MHz) instruments. ESI-FTMS spectra were measured on a
Bru¨ker-Magnex BioAPEX 30es ion cyclotron high-resolution
HPLC-FT spectrometer. HPLC was performed on a Waters LC
Module 1, using a Phenomenex Prodigy reversed-phase (150
× 4.6 mm, 5 µm) for screening and a Phenomenex Prodigy
reversed-phase (250 × 10 mm, 10 µm) for preparative work;
UV detection was achieved at 203 nm. Optical rotations were
measured on a J ASCO DIP 370 Digital Polarimeter.
Isola tion . The stems from C. retusa L. (Rhamnaceae) were
collected in Venezuela; a Voucher specimen (# V13021) was
deposited at the National Center for Natural Products Re-
search, University of Mississippi. Air-dried stems (500 g) were
ground to a coarse powder and extracted at 37° with 95% EtOH
(2.5 L × 4, 3 h, each). The EtOH extract was suspended in
water (1.5 L) and extracted with CHCl3 (3 × 1 L) and then
with 1-BuOH (3 × 1 L). The combined BuOH fractions were
evaporated to dryness in-vacuo at 45° to afford a yellow residue
(5.9 g). Part of the residue (5.0 g) was subjected to column
chromatography on silica gel using a mobile phase consisting
of CHCl3/MeOH/H2O mixtures starting at 70:10:1 and ending
at 20:10:1(6 L). Fractions of 25 mL each were collected, spotted
on TLC, and combined according to TLC similarities. A total
Ack n ow led gm en t. The authors thank Dr. J ames M.
O’Neal for assistance in obtaining the HRESI-MS spectra. This
work was supported by the National Institute of Allergy and
Infectious Diseases, DAIDS, NIH, Bethesda, MD, Grant No.
AI 27094.
Refer en ces a n d Notes
(1) Heywood, V. H., Ed. Flowering Plants of the World; Oxford University
Press: 1993; pp 187-188.
(2) Li, X.-C.; ElSohly, H. N.; Nimrod, A. C.; Clark, A. M. J . Nat. Prod.
1999, 62, 674-677.
(3) Higuchi, R.; Kubota, S.; Komori, T.; Kawasaki, T.; Pandey, V. B.;
Singh, J . P.; Shah, A. H. Phytochemistry 1984, 23, 2597-2600.
NP9901940