Journal of Natural Products
Article
was obtained from fraction D3, 2 (100 mg), 3 (25 mg), and 4 (15 mg)
were obtained from fractions D8, D11, and D13, respectively, D15
(4.7 mg) gave a mixture of 5 and 6, L-chiro-inositol9 (150 mg)
crystallized from fraction F, fraction G (30 mg) was subjected to silica
gel TLC eluting with n-BuOH/2-propanol/formic acid/H2O
(48:17:18:17) to give chlorogenic acid8 (15 mg), and astragalin7 (25
mg) was isolated from fraction I (67 mg) in the same way.
was established as ent-15β-(β-L-fucosyloxy)kaur-16-en-19-oic
acid.
A minor amount of a mixture of 5 and 6 (4.5 mg) was
1
isolated. Their H and 13C NMR data (Table 2 for 5 and
Experimental Section for 6) were similar to those of 2 and 3,
respectively, except for the absence of the glucose moiety
resonances at C-19. Although 5 and 6 were isolated as natural
products, they could not be fully identified. Thus, enzymatic
hydrolysis of a mixture of 1−3 using β-glucosidase from
almonds gave a sufficient quantity of 5 for its complete
characterization, as shown in the Experimental Section and in
Table 2.
ent-15β-(β-L-Fucosyloxy)kaur-16-en-19-oic acid β-D-glucopyra-
nosyl ester (1): white powder; mp 145−150 °C; [α]25 −31.6 (c
D
0.01, MeOH); IR (KBr) νmax 3370, 2924, 2856, 1732, 1655, 1061
cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z [M + Na]+
649.3222 (calculated for C32H50O12 + Na, 649.3200).
ent-15β-(4-Acetoxy-β-L-fucosyloxy)kaur-16-en-19-oic acid β-D-
glucopyranosyl ester (2): white powder; mp 150−152 °C; [α]25
D
The antiprotozoal activity of 1−4 was tested on Entamoeba
histolytica and Giardia lamblia trophozoites (Table 5). In
−46.6 (c 0.01, MeOH); IR (KBr) νmax 3373, 2925, 2857, 1724, 1655,
1602, 1240, 1070 cm−1; 1H and 13C NMR, see Table 1; HRESIMS m/
z [M + Na]+ 691.3316 (calculated for C34H52O13 + Na, 691.3306).
ent-15β-(3-Acetoxy-β-L-fucosyloxy)kaur-16-en-19-oic acid β-D-
Table 5. In Vitro Antiprotozoal Activity of Compounds 1−4
glucopyranosyl ester (3): white powder; mp 145−148 °C; [α]25
D
a
IC50 μM (CI)
−33.0 (c 0.01, MeOH); IR (KBr) νmax 3691, 3597, 2929, 1856, 1734,
1602, 1022 cm−1; 1H and 13C NMR, see Table 2; HRESIMS m/z [M
+ Na]+ 691.3316 (calculated for C34H52O13 + Na, 691.3306).
ent-15β-(β-L-Fucosyloxy)kaur-16-en-19-oic acid (4): white pow-
compound
Entamoeba histolytica
Giardia lamblia
1
2
3
4
43.3 (43.6−43.1)
49.5 (50.1−49.0)
52.7 (52.9−52.3)
73.5 (73.9−72.8)
2.18 (2.2−2.14)
0.23 (0.58−0.17)
41.9 (42.2−41.5)
69.5 (69.9−69.2)
48.9 (49.3−48.7)
98.5(98.7−97.8)
0.83 (0.87−0.82)
1.22 (1.57−0.81)
der; mp 142−146 °C; [α]25 −36.7 (c 0.01, MeOH); IR (KBr) νmax
D
3368, 1731, 1706, 1655, 1231 cm−1; 1H and 13C NMR data, see Table
2; HRESIMS m/z [M + Na]+ 487.2667 (calculated for C26H40O7 +
Na, 487.2672).
c
emetine
c
metronidazole
ent-15β-(4-Acetoxy-β-L-fucosyloxy)kaur-16-en-19-oic acid (5):
white powder; mp 149−152 °C; [α]25 −28.0 (c 0.01, MeOH); IR
a
D
Results are expressed as mean (n = 6), CI = 95% confidence intervals;
1
(KBr) νmax 3596, 1735, 1693, 1449, 1370, 1242 cm−1; H and 13C
c
correlation coefficient >0.9500 and p < 0.05. Positive controls.
NMR data, see Table 2; DART-MS m/z [M + H]+ 507.
ent-15β-(3-Acetoxy-β-L-fucosyloxy)kaur-16-en-19-oic acid (6): 1H
NMR partial data extracted from a 5 and 6 mixture (pyridine-d5, 500
MHz) δ 2.61 (1H, s, H-13), 5.88 (1H, s, H-17a), 5.11 (1H, s, H-17b),
1.32 (3H, s, CH3-18), 1.21 (3H, s, CH3-20), 1.96 (3H, s, OCOCH3),
4.92 (1H, d, J = 7.6 Hz, H-1′), 4.65 (1H, dd, J = 10.0, 7.6 Hz, H-2′),
5.46 (1H, dd, J = 10.0, 3.2 Hz, H-3′), 4.28 (1H, d, J = 4.0 Hz, H-4′),
3.88 (1H, qd, J = 6.4, 0.8 Hz, H-5′), 1.58 (3H, d, J = 6.4 Hz, CH3-6′);
13C NMR (CDCl3, 125 MHz) δ 41.1 (CH, C-13), 106.6 (CH2, C-17),
29.7 (CH3, C-18), 180.7 (C, C19), 16.6 (CH3, C-20), 171.2 (C,
OCOCH3), 21.4 (CH3, OCOCH3), 107.1 (CH, C-1′), 72.8 (CH, C-
2′), 78.6 (C, C-3′), 70.5 (CH, C-4′), 71.6 (CH, C-5′), 17.6 (CH3, C-
6′).
general, all compound showed moderate activity against both
protozoa with IC50 values ranging from 43.3 to 73.5 μM for E.
histolytica and from 41.9 to 98.5 μM for G. lamblia.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
measured on a Fisher-Johns apparatus and are uncorrected. IR spectra
were obtained on a Bruker Tensor 27 spectrometer. VCD data were
acquired on a BioTools dualPEM ChiralIR FT-VCD spectropho-
tometer (Jupiter, FL, USA). The 1D and 2D NMR experiments were
1
performed on a Bruker Advance III spectrometer at 400 MHz for H
Paniculoside V (7): white powder; mp 168−172 °C (reported6
173−175 °C); 13C NMR data were essentially the same as reported;6
1H NMR (pyridine-d5, 400 MHz) δ 1.82 (1H, d, J = 12.4, H-1a), 0.83
(1H, td, J = 12.4, 3.2, H-1b), 2.19 (1H, m, H-2a), 1.38 (1H, m, H-2b),
2.34 d (1H, d, J = 12.8, H-3a), 0.95 (1H, dd, J = 13.6, 4.4, H-3b), 1.30
(1H, m, H-5), 2.43 (1H, m, H-6a), 2.08 (1H, m, H-6b), 2.44 (1H, m,
H-7a), 1.48 (1H, m, H-7b), 1.92 (1H, m, H-9), 1.90 (1H, m, H-11a),
1.43 (1H, m, H-11b), 1.62 (1H, m, H12a), 1.46 (1H, m, H-12b), 2.57
(1H, br s, H-13), 2.22 (1H, m, H-14a), 1.00 (1H, m, H-14b), 4.11
(1H, t, J = 2.4, H-15), 5.89 (1H, br s, H-17a), 5.09 (1H, dd, J = 1.6,
0.8, H-17b), 1.24 (3H, s, CH3-18), 1.29 (3H, s, CH3-20), 5.04 (1H, d,
J = 7.6, H-1′), 4.12 (1H, t, J = 8.8, H-2′), 4.27 (1H, m ov, J = 9.2, H-
3′), 4.25 (1H, m ov, H-4′), 3.92 (1H, ddd, J = 8.8, 4.8, 2.8, H-5′), 4.54
(1H, dd, J = 11.2, 2.8, H-6′a), 4.42 (1H, dd, J = 12.0, 4.0, H-6′b), 6.27
(1H, d, J = 8.0, H-1″), 4.21 (1H, t, J = 8.8, H-2″), 4.28 (1H, t, J = 8.4,
H-3″), 4.34 (1H, t, J = 9.2, H-4″), 4.03 (1H, ddd, J = 9.2, 4.4, 2.4, H-
5″), 4.46 (1H, dd, J = 12.0, 2.4, H-6″a), 4.38 (1H, dd, J = 12.0, 4.8, H-
6″b).
Methyl xylopate (8): A solution of 4 (17 mg) in H2O (2.0 mL) was
treated with 23 mg of NaIO4 and stirred at rt for 18 h. Then, 20 mg of
KOH was added and the reaction mixture refluxed for 1 h. After
addition of H2O (2.0 mL) the pH was adjusted to 4−5 with HOAc,
and the resulting mixture was extracted with EtOAc (3 × 10 mL). The
EtOAc fraction was washed with H2O (25 mL), dried over anhydrous
Na2SO4, and evaporated under reduced pressure to yield 9, which
without purification was subjected to esterification with diazomethane,
followed by acetylation with pyridine and Ac2O to give 8 (1 mg),
and 100 MHz for 13C and on a Varian Unity Inova 500 MHz for H
1
and 125 MHz for 13C. Chemical shifts were referenced to TMS, and J
values are given in Hz. The HRESIMS data were recorded on a Waters
Synap G2S HDMS Q-TOF mass spectrometer. The DART-MS data
were obtained on a Jeol AccuTOF JMS-T100LC mass spectrometer.
Preparative TLC was carried out on precoated Macherey Nagel Sil G/
UV254 plates of 1.0 mm thickness. Silica gel 230−400 mesh
(Macherey-Nagel), Sephadex LH-20 (Pharmacia Biotech), and
octadecyl-functionalized silica gel (Sigma-Aldrich) were used for
column chromatography.
Plant Material. A. cylindrica was collected from Tenancingo, State
of Mexico, Mexico, in February 2012. The plant material was identified
by Jose Luis Villasenor, and a voucher specimen (MEXU-1333 472)
̃
was deposited at the National Herbarium (MEXU) of the Instituto de
́
Biologia, UNAM, Mexico City, Mexico.
Extraction and Isolation. The dried and powdered leaves of A.
cylindrica (100 g) were added to boiling H2O (1 L × 3) and filtered
immediately. The infusion was filtered, and after cooling to room
temperature it was lyophilized to yield 33 g of residue. This material
was fractionated over a Sephadex LH-20 column, using MeOH as
eluent, to give nine fractions (A−I). Fraction D (950 mg) was
subjected to flash column chromatography on octadecyl-functionalized
silica gel, with an isocratic mobile phase of MeOH/H2O (1:3, v/v)
with a 5.0 mL min−1 flow rate, to obtain 50 fractions (25 mL each),
which were combined into 15 major fractions (D1−D15) by TLC
evaluation. D1 (35 mg) was subjected to silica gel CC eluting with
EtOAc/MeOH/H2O (200:16:7), affording 7 (25 mg). Pure 1 (30 mg)
F
J. Nat. Prod. XXXX, XXX, XXX−XXX