778
BONACHEVA, BOTIROV
Based on comparison with authentic kaempferol conclude the hydroxyl group at the position 7 is substiꢀ
by TCL analysis in the chloroform–ethyl acetate solꢀ tuted for Lꢀrhamnose while 3ꢀOH is glycosylated by
vent system (6 : 3 : 1), the spectral data and physicoꢀ Dꢀgalactose.
chemical properties compound 1 was determined as
3,5,7,4'ꢀtetrahydroxyflavon (kaempferol) [12, 13].
Kaempferol from Equisetum silvaticum L. was isolated
for the first time.
The signal of anomeric proton of Dꢀgalactose in
the 1H NMR spectrum (DMSOꢀd6) was observed as a
doublet with the spinꢀspin coupling constant of 7.3 Hz
that indicates that Dꢀgalactose is in ꢀconfiguration.
β
On the basis of the chemical conversion and comꢀ
parison of the spectra with the literature data we idenꢀ
O
R2O
OH
tified compound 2 as kaempferol 3ꢀ
sylꢀ7ꢀ ꢀrhamnopyranoside [14]. Kaempferol 3ꢀ
Dꢀgalactosylꢀ7ꢀ ꢀrhamnopyranoside was isolated
from Equisetum L. plants for the first time.
Kaempferol 3ꢀ ꢀrutinosylꢀ7ꢀ ꢀrhamnopyranoꢀ
O
ꢀ
β
ꢀDꢀgalactoꢀ
OR1
O
Oꢀβꢀ
OH
O
O
1. R1 = H, R2 = H
Oꢀ
β
O
side (3) is a lightꢀyellow crystalline substance with the
2. R1 =
3. R1 =
β
ꢀDꢀGalp, R2 =
α
ꢀLꢀRhap
molecular formula of C33H40O19, the melting point of
β
ꢀDꢀGlcp)6 1)ꢀ
←
α
ꢀLꢀRhap, R2 = ꢀLꢀRhap.
α
147–148
C, and the mass spectrum (m/z): 286 (M+ of
°
Kaempferol 3ꢀOꢀ
rhamnopyranoside (2) is a yellow crystalline comꢀ
pound with the molecular formula of C27H30O15, the
melting point of 188–190 C, and the mass spectrum
β
ꢀDꢀgalactopyranosylꢀ7ꢀOꢀ ꢀLꢀ
α
aglycone). The UV spectrum: λmax (ethanol) 272 and
361 nm; + NaOH: 277 and 400 nm; +AlCl3: 276, 350,
and 402 nm; +CH3COONa: 267 and 356 nm.
°
The IR spectrum: OHꢀgroups (3373–3277 cm–1),
(m/z): 286 (M+ of kaempferol aglycone). The UV
spectrum: λmax (ethanol): 272 and 359 nm; + NaOH:
277 and 399 nm with the decreased intensity of band I;
+AlCl3: 276, 348, and 400 nm; +CH3COOH: 267 and
354 nm. On the basis of UV spectra we concluded that
the substance belongs to 3,7ꢀdiꢀOꢀsubstituted flaꢀ
vonols.
γ
ꢀpyrone C=O (1654 cm–1), aromatic C=C (1591 cm–1)
and glycoside CꢀO (1100–1000 cm–1) bonds, etc.
1H NMR spectrum (
, DMSOꢀd6): 0.98 (d, 6.95 Hz,
δ
CH3 of rhamnose biose), 1.13 (d, 6 Hz, CH3ꢀ7ꢀRha),
3.05–3.87 (m, protons of the sugar part), 5.16 (d, 2.1
Hz, Hꢀ1''' anomer of HꢀRha biose), 5.32 (d, 7.5 Hz, Hꢀ1'''
anomer of HꢀGlc), 5.53 (d, 2 Hz, Hꢀ1'' of HꢀRha),
6.42 (H, d, 2.0 Hz, Hꢀ6), 6.76 (H, d, 2.0 Hz, Hꢀ8), 6.87
(2H, d, 8.8 Hz, Hꢀ3', Hꢀ5'), 8.00 (2H, d, 8.8 Hz, Hꢀ2',
Hꢀ6'), 12.6 (bs, 5ꢀOH).
The complete acid hydrolysis of glycoside 3 by the
mixture of 5% HCl and ethanol (1 : 1) resulted in
kaempferol (4 mg), Lꢀrhamnose, and Dꢀglucose.
The IR spectrum of substance 2 contains the
absorption bands of hydroxyl groups (3392–3219 cm–1),
γ
ꢀpyrone carbonyl (1647 cm–1), glycoside CꢀO
(1006–1141 cm–1) and aromatic bonds.
1H NMR (
, DMSOꢀd6): 12.6 (1H, bs, 5ꢀOH),
β
8.06 (2H, d, 8.8 Hz, Hꢀ2',6'), 6.95 (2H, d, 8.8 Hz,
Hꢀ3',5'), 6.80 (1H, d, 1.9 Hz, Hꢀ8), 6.43 (1H, d, 1.9 Hz,
Hꢀ6), 5.53 (s, Hꢀ1'' anomer of HꢀRha), 5.47 (d, 7.3 Hz,
Hꢀ1''' anomer of HꢀGal), 3.05–3.62 (m, protons of
the sugar part), 1.05 (d, 6 Hz, CH3ꢀRha).
In order to reveal the positions of carbohydrate resꢀ
idues we performed alkaline and mild stepwise acid
hydrolysis of substance 3. A diglycoside which was
identified as kaempferol 3ꢀOꢀglucosylꢀ7ꢀOꢀrhamnoꢀ
Substance 2 (14 g) underwent complete acid
hydrolysis by a mixture of 5% HCl–C2H5OH (1 : 1) in
a water bath for 2 h. The aglycone precipitate formed
during ethanol distillation under vacuum was sepaꢀ
rated by filtration and recrystallized. It resulted in
kaempferol of the C15H10O6 composition (λmax 266,
side (melting point of 151–153
°
C; UV spectrum: λmax
272 and 359 nm) and a monosaccharide Lꢀrhamnose
were formed at the first stage (after 15 min) of mild
acid hydrolysis [4, 8]. A glycoside (melting point of
177–179 C; UV spectrum: λmax 260 and 366 nm) and
°
Dꢀglucose are formed at the second stage (after 30 min)
(TLC, IR spectrum). We identified the glycoside as
369 nm; melting point of 270–272 C). The filtrate was
°
evaporated to dryness; the precipitate was dissolved in
ethanol. Its TLC analysis in the nꢀbutanolꢀacetic acidꢀ
water solvent system (6: 1.5 : 2.5) in the presence of
authentic samples revealed Dꢀgalactose and Lꢀrhamꢀ
nose.
kaempferol 7ꢀOꢀrhamnoside by comparison with the
literature data [15–16].
The alkaline hydrolysis of compound 3 resulted in
a monosaccharide Lꢀrhamnose and the substance with
the melting point of 182–185 C and the UV spectrum:
°
We also performed alkaline hydrolysis of 10 mg of λmax 266 and 352 nm [4]. The comparison with the litꢀ
substance 2 by 0.5% KOH (10 mL) in a water bath for erature data allowed us to identify the substance as
2 h. It resulted in kaempferol 3ꢀ
Oꢀ
β
ꢀDꢀgalactopyraꢀ kaempferol 3ꢀOꢀrutinoside which had been isolated
noside and Lꢀrhamnose (TLC). Consequently, we can from the wood horsetail earlier. The sugar unit was
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 7
2014