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A.P. Singh et al. / Phytochemistry 63 (2003) 485–489
3.2. Plant material
(500 MHz, CDCl3): ꢀ 5.61 (1H, d, J=3.5Hz, anomeric
–H), 5.38 (1H, d, J=5.5Hz, –CHOAc), 5.30 (1H, t,
J=6.0 Hz, ꢀCHOAc), 5.00 (1H, t, J=11 Hz,
–CHOAc), 4.80 (1H, dd, J=10.0 and 4.0 Hz, –CHOAc),
4.04–4.30 (8H, m, 3ꢂ–CH2OAc, 2ꢂCHO), 2.12 (3H, s,
ꢀOCOCH3), 2.01–2.05 (15H, s, 5ꢂ–OCOCH3), 1.94
(3H, s, ꢀOCOCH3), 1.92 (3H, s, ꢀOCOCH3).
The epigeal part of Petunia nyctaginiflora Juss were
collected from the campus of Banaras Hindu Uni-
versity, Varanasi. A specimen of the plant material has
been preserved in the Department of Medicinal Chem-
istry, IMS, Banaras Hindu University, Varanasi, India.
3.3. Extraction and isolation
3.6. Benzoylation of PNB
Air-dried epigeal part of Petunia nyctaginiflora (5 kg)
was milled and soxhletted with n-hexane. The n-hexane
extract (20 l) was concentrated to 2.5 l and partitioned
against MeOH–H2O (1:4) mixture. The aqueous MeOH
soluble portion obtained was evaporated under reduced
pressure to a residue (19.5 g), which was chromato-
graphed over silicagel (60–120 mesh) column and eluted
with solvents of increasing polarity. C6H6–EtOAc (4:6)
eluates yielded PNB (7 g).
IR( ꢁmax) 3600, 3510 and 1745 cmꢀ1.1H NMR(500
MHz; CDCl3): ꢀ 5.65 (1H, d, J=3.5 Hz, anomeric-H),
5.62 (1H, m, –CHOR), 5.15 (1H, m, –CHOR), 4.96 (1H,
dd, J=10.2 and 3.9 Hz, –CHOR), 4.20 (4H, m), 3.78
(2H, m), 3.45 (4H, m, 2ꢂ–CH2OH), 2.30 (m, –COCH2),
1.25 (br, methylenes), 1.10–0.98 (doublets, secondary
–CH3).
A mixture of PNB (0.2 g), benzoyl chloride (2 ml),
pyridine (0.5 ml) was kept overnight under anhydrous
conditions. The reaction mixture was poured over cru-
shed ice, stirred for 0.5 h and then extracted with
CHCl3, washed, dried, freed from pyridine in a vacuum
and chromatographed over silicagel. Elution with
n-hexane–EtOAc (8:2), yielded PNB benzoate (0.25 g).
3.7. Isolation of PNBbenzoate I ( 1) and PNBbenzoate
II (3)
PNB benzoate (0.21 g) was dissolved in MeOH (2 ml)
and was purified by reverse phase Shim-pack silicagel
column (RP—18). The detector used was UV (254 nm),
flow rate was 6 ml/min., with MeOH as eluant. HPLC
chromatogram showed two prominent peaks along with
several small peaks. The fractions corresponding to
peaks showing retention time 19.3 min and 21.5 min
were collected separately and designated as PNB
benzoate I (1) and PNB benzoate II (3) respectively. The
solvent was removed under reduced pressure. PNB
benzoae I (1): C68H78O19; 1H NMR(270 MHz, CDCl 3):
ꢀ 8.04 (10H, m), 7.45 (15H, m) 6.0 (1H, d), 5.92 (1H, m),
5.85 (1H, m), 5.15 (1H, m), 4.95 (1H, dd), 4.63 (3H, m),
4.36 (1H, dd), 4.08 (2H, br), 2.15 (6H, t), 1.53 (3H, br
m), 1.25 (12H, br s), 1.12 (18H, d, J=6.7 Hz).
3.4. Acetylation of PNBto PNBacetate
A mixture of PNB (0.2 g), Ac2O (1 ml) and pyridine
(0.5 ml) was kept overnight under anhydrous conditions
at room temperature. The reaction mixture after usual
work up yielded PNB penta acetate (0.22 g) as a gummy
residue.
1H NMR(500 MHz, CDCl ): ꢀ 5.92 (3H, m), 5.22
3
(1H, d, J=3.5 Hz, anomeric –H), 5.1 7 (1H, m), 4.86
(1H, dd, J=10.2 and 3.9 Hz), 4.30 (3H, m), 4.15 (5H,
m), 2.17 (6H, s), 2.14 (3H, s), 2.09 (3H, s), 2.07 (3H, s),
1.22 (br s, methylenes), 1.08 (d, methyls).
PNB benzoae II (3): C71H84O19; 1H NMR(270 MHz,
CDCl3): ꢀ 8.05 (10H, m), 7.45 (15H, m), 6.01 (1H, d),
5.92 (1H, m), 5.85 (1H, m), 5.16 (1H, m), 4.93 (1H, dd),
4.63 (3H, m), 4.36 (1H, dd), 4.07 (2H, br), 2.15 (6H, t), 1.50
(3H, br m), 1.25 (18H, br s), 1.12 (18H, d, J=6.7 Hz).
3.5. Basic hydrolysis of PNB
A solution of PNB (0.2 g) in 10 ml of 5% methanolic
KOH was refluxed for 1 h. The basic solution was
cooled, neutralized carefully with 2 N HCl and diluted
with water (10 ml). The resultant solution was extracted
with Et2O (3ꢂ10 ml) and then MeOH was evaporated
under reduced pressure. The solution was then extracted
with n-BuOH. The n-BuOH soluble portion was evapo-
rated to dryness. The residue obtained was divided into
two parts. Part I was crystallised from aqueous alcohol
to colourless cubes; C12H22O11; mp 183–185 ꢁC;
[a]D+66.1ꢁ (H2O). Part II was left overnight with
Ac2O/pyridine at room temperature. The usual work up
of the reaction mꢁixture yieldedꢁ sucrose octa acetate,
C28H38O19: mp 88 C; [a]D+58.3 (C2H5OH). 1H NMR
3.8. Basic hydrolysis of PNB benzoate I
Basic hydrolysis of PNB benzoate I (1) was carried
out as described in case of PNB. The sugar part
obtaꢁined was identified as sucrose, C12H22O11, mp 183–
185 C, while the Et2O soluble portions were dried and
evaporated to give acid mixture.
3.9. p-Bromophenacyl ester of acids
The acetonic solution of the acid mixture (5 mg in 2
ml) obtained from the basic hydrolysis of 1 was added
to a solution of p-bromophenacyl bromide (27.8 mg)
and triethylamine (2 drops) in acetone (2 ml). The