Indian Journal of Chemistry
Vol. 438, January 2004, pp. 223-226
Note
Isolation and characterization of selected secondary metabolites from dry leaves of Quercus semicarpifolia.
S M S Chauhan *, Mamta Singh & Anil Kumar
Department of Chemistry, University of Delhi , Delhi 110007.
India
Email : [email protected] .ac .in
Received 21 April 2003; accepted (revised) 19 AlIglist 2003
Nonacosane, friedelin 1, ~- s it o sterol 2 and oleanolic acid 3
from petroleum ether; 5-acetoxy-3 ,3',4',5' -tetramethoxyflavone 4
from acetone and quercetin 5 from methanolic extract of the dry
leaves of Querclls selllicarpiJolia have been isolated .
Quercus semicarpijolia (Kharshu oak) is one of the
major food-plant of temperate tasar silkworm Alltheraea pernyi. Quercus are rich sources of varying polyphenolic compounds including hydrolysable and condensed tannins. I-4 The condensed tannins and related
phenolics form complexes with proteins and these
interactions have been correlated with feeding, growth
and survival of herbivores. s-Io Phenolics are important
secondary metabolites for constitutive and induced
defense against herbivores and pathogens.s.6 The tannins of oak leaves inhibit the hydrolysis of food proteins by trypsin. I1.1 2
The secondary metabolites such as friedelin 1, ~­
sitosterol 2, taraxerol, quercetin 4, quercetin 3-0-1rahmnoside, pelarogonidin-3-D-glucoside and quercetin 3-0-disaccharides have been isolated from bark
of Quercus semicarpijolia, 13. 14 but no detailed investigation of secondary metabolites from dry leaves of
Quercus semicarpijolia have been examined so far.
Herein, we report for the first time the isolation and
characterization of secondary metaboLtes from the
petroleum ether, acetone and methanol extract of dry
leaves of Quercus semicarpijolia with a view to understa~ding their role in the feeding and fecundity
behaVIOr of temperate tasar silkworm Alltheraea
pemyi.
Results and Discussion
The secondary metabolites act as feeding attractant
and .deterrents ~nd also affect the quality and quantity
of silk productIOn from silkworms. They also provide
biochemical defense to plants.s.6 The f1avonoids are
large group of secondary metabolites from higher
plants and are responsible for flavor in foods and colour in flowers. IS
The nonacosane, friedelin 1, ~-sitosterol 2 and
oleanolic acid 3 have been isolated from petroleum
ether extract of Quercus semicarpijolia and the structure of the compounds have been elucidated with different chemical tests, m.p., JR, IH NMR and EJ-MS
spectroscopic data (see experimental).
A new compound 5-acetoxy-3,3',4',5'-tetramethoxyflavone 4 has been isolated from acetone extract of dry leaves of Quercus selllicarpijolia. Structure of 4 has been confirmed by various spectroscopic
techniques and acidic hydrolysis of 4 to corresponding 5-hydroxy derivative 5 (see experimental) . Positive ferric chloride test and the presence of a singlet at
8 12.51 ppm in IH NMR spectrum of hydrolyzed
product 5 confirms the position of hydroxy group at
C-5 carbon chelated with carbonyl at position 4.
Quercetin 6 has been isolated from methanolic extract of the dry leaves Quercus semicarpijo/ia. It gives
blue colour with ferric chloride and red colour with
vanillin-HCI, which indicates the presence of flavonoid moiety. The structure of 6 has been confirmed by
NMR, IR, UY-visible and EI-MS spectroscopic data
(see experimental).
Experimental Section
The IHand I3C NMR spectra were recorded on a
Bruker Heaven (300 MHz) spectrophotometer using
TMS as internal standard (chemical shifts in 8, ppm).
The electron impact mass spectra were recorded on a
Jeol SX 102/ DA (KY 10 mA) instrument. lR spectra
were recorded in KBr on a Perkin-Elmer spectrum
IT-2000 spectrometer. The electronic spectra were
recorded on a Shimadzu UY-260 spectrophotometer
an? the Amax were expressed in nanometer. All melting
POll1lS were determined on a Thomas Hoover Unimclt
melting point apparatus and are uncorrected. Dry
leaves of Quercus semicarpijolia were collected from
Palampur (Himachal Pradesh).
Isolation of different compounds from petroleum ether extract of dry leaves of Quercus semicarpifolia. The powdered dry leaves (600 g) of Quer-
224
INDIAN J. CHEM ., SEC B. JANUARY 2004
o
110
Friedelin 1
f)-Sit osterol 2
OCH.1
HO
Oleanolic acid 3
5-Acetox y-3',3',4' ,5' -tetramethox y fla vone 4
OH
OCH ~
~
0
~
OCH ~
~
HO
#
OCH ~
OH
OCH,.>
OH
OH
0
5-Hyclroxy-3.3,4,5-letramethoxyflavone 5
CIIS selll icarpijo/ia were extracted with petroleum
ether us ing soxhlet for 48 hr, the solvent was removed
under reduced pressure and the residue (2.0 g) was
chromatographed over sil ica gel (60- 120 mesh , 10.0
g) column . Elution of column with petroleum ether
gave 80 mg of a white compound . The physical and
spectroscopic data indicated that the compound to be
nonacosane. R,.: 0.64 (C 6 H(,) ; 64-6°C (Iit. 16 m.p.
66°C); UV (CHC I" AmaJnm) : 212.57 , 272.2 1; IR
(KBr/cm· I): 2850, 1653, 1542 and 1464; IH NMR
(CDCI ]): 8 0.87 (t, 6H, CH]), 1. 18-1 .56 (m. 54H.
CH 2-protons) ; LiC NMR (CDCI 3 ): 8 14.54, 18 .52,
20.13,29.31 , 29.56, 29.67,29.75 , 30.40,30.83,30.98,
31.66,31 .78,31 .89, 32.33 and 32.70.
Elution of column with petrol eum ether: benzene
(60: 40 v/v) furnished 25 mg of a compound, which
OH
0
Quercetin 6
gave positive Lieberman-Burchard test for terpenoids
and positive DNP test for carbonyl group . The physical and spectral data indicated the compound to be
friedel in 1. R,.: 0.47 (C 6 H6); 258-60°C (lit. l ? m.p. 25561 °C) ; UV (CHCI" AmaJnm): 217 .43, 245.52 ; IR
(KBr/cm' I): 2925, 1714 (C=O), 1458, 1375 and 1120;
IH NMR (CDCI 3 ): 80.72 (s, 3H , C 24 -CH, ), 0.87 (6H ,
C 25-CH , & Cn -CH, ), 0.95 (s, 6H , C:IO-CH, & C 2'r
CH]), 1.00 (s, 6H , C 26 -CH 3 & C 27 -CH, ), 1.06 (s, 3H ,
C 2s-CH 3) , 1.18-1.56 (m, 22H), 1.94 (m, I H , CI-H axial). 2.22 (q, I H , C 4 -H), 2.25 (m. 2H. C 2-H): I3C
NMR (CDCl, ): 8213 . 13 (C-3), 59. 52 (C- IO), 58. 26
(C-4), 53.13 (C-8), 42.84 (C-18), 41.54 (C-2), 41.33
(C-6), 39.73 (C-13), 39.27 (C-22), 37.48 (C-14),
36.04 (C-9), 35.66 (C- 16), 35.37 (C-I I), 35.03 (C19),32.81 (C-29), 32.45 (C-21), 32. 1 I (C-15) , 3 1.79
NOTES
(C-28), 30.52 (C-30), 30.02 (C-12), 29.71 (C-17),
28.18 (C-20), 22.29 (C-l), 20.27 (C-5), 18.66 (C-27),
18 .26 (C-7), 18.02 (C-26), 17.95 (C-25), 14.67
(C-24), 6.82 (C-23) ; EI-MS (mlz): 426 (28),411 (10),
393 (7), 218 (l00), 189 (70), 175 (25), 137 (65) and
109 (37) .
Further elution of column with petroleum ether:
benzene (30: 70, v/v) gave 23 mg of ~-sitosterol 2 with
following physical and spectral data. R r: 0.32 (C 6 H6);
136-38°C (lit. IS m.p. l38-39°C); IR (KEr/cm- I): 3400,
3000,2980, 1640, 1400 and 1050; IH NMR (COCl) : 8
0.68 (s, 3H, Ci s-CH,), 0.8-0.99 (m, 15H, 5 x CH3),
1.21-1.32 (m, 2IH), 1.35-1.37 (m, 4H), 3.56 [m, IH, CH(OH)], 5.35 (m, I H, C=CH); EI-MS (mlz): 414
(100), 396 (10), 368 (22), 350 (40), 229 (48), 270 (53),
254 (65), 158 (45), 81 (62) and 55 (78).
Finally, elution of column with benzene gave 28 mg
of oleanolic acid 3 with following physical and spectral
data. Rr: 0.47 (C 6 H6) ; 300-02°C (lit. 19 m.p. 298-300°C) ;
IR (KEr/cn,-I) : 3400, 1720, 1490, 1020; IH NMR
(COCI ) : 8 0.64 (s, 3H, C WCH3), 0.78-1.09 (m, 18H, 6
x CH 3) , 1.13-1.35 (m, 19H), 1.41-1.46 (m, 4H), 3.32
(brs, I H, -OH), 3.56 [m, I H, -CH(OH)], 5.35 (m, 1H,
C=CH), 11.56 (s, IH -COOH); EI-MS (mlz): 457 (10),
248 (45),208 (15), 207 (70), 203 (50).
The structure of oleanolic acid 3 was further confirmed by preparing its methyl ester with diazomethane in diethyl ether. To a suspension of 3 (10 mg) in
dry diethyl ether (10 mL) ethereal solution of diazomethane was added drop wise and reaction mixture
was stirred at room temp for 2 hr. Oiethyl ether was
removed under reduced pressure carefully and the
residue was crystallized from acetone. R r: 0.32
(C 6 H6) ; 196-99°C (lit. 20 m.p. 199-200°C); IR
(KBlkm-I): 3200, 2757, 1820, 1740 and 1020; IH
NMR (COCh): 80.66 (s, 3H, C ls-CH 3), 0.81-1.04 (m,
18H , 6 x CH.,), 1.11-1.37 (m, 19H), 1.38-1.43 (m,
4H), 3.26 (brs, IH , -OH), 3.38 [m, IH , -CH(OH)],
3.47 (s, 3H, OCH 3), 5.45 (m, IH, C=CH); EI-MS
( mlz) : 469 (2.9), 411 (1.3), 409 (2 . 1), 264 (1.1), 262
(51.7), 203 (100), 202 (21.9) & 157 (2. 1). The m.p.
and spectral data indicated the compound to be
methyl oleanolate.
Isolation of 5-acetoxy-3,3' ,4' ,5'-tetramethoxyflavone 4 from acetone extract of dry leaves of
Quercus semicarpifolia. The organic residue left after
petroleum ether extract of Qu ercus selllicarp!folia
was extracted with acetone for 60 hr using soxhlet.
The extracted mixture was concentrated under reduced pressure and the residue was chromatographed
225
over silica gel (60-120 mesh) column. Elution of column with chloroform: methanol (80 : 20) gave 40 mg
of a white coloured compound. It gave positive vanillin-HCI test, which indicate the presence of flavonoid
moiety. Rr: 0.23 (CHCI 3); m.p. 216-18°C; UV
(CHJOH, Amax/nm): 338.2, 252.6 and 223; IR
(KBr/cm- I): 2927, 1766.7, 1627, 1604, 1507, 1473 ,
1380, 1191,1128 and 1009; IH NMR (COCl ) : 8 2.48
(s, 3H, -OCOCH3), 3.82 (s, 3H, C 3-OCH 3 ) , 3.94 (s,
9H, 3 x OCH3), 7.00 (d, 1=7.4 Hz, IH, Cg-H), 7 .37 (s,
2H, C 2 ·.6 ·-H ), 7.44 (d, 1=8.26 Hz, C 6 -H), 7.64 (t, IH,
CrH); I)C NMR (OMSO-d6 ) : 21.67 (OCOCH 3 ) ,
56.43 (CJ'.5,-OCH 3), 60.43 (C 4 ,-OCH 3), 60.99 (C r
OCH 3), 106.90 (C 2'.6'), 117.29 (C s), 117.49 (C IO),
119.47 (C 6 ) , 125.95 (C I'), 134.47 (C 7 ) , 140.48 (C 9 ),
141. 7 (C 4') , 149.40 (C n ,), 153.64 (C 2), 154.66 (C s),
156.57 (C 3), 169.68 (OCOCH) and 173.53 (C 4 ); ElMS (mlz): 400 (100), 384 (12), 326 (100), 297 (52) ,
281 (59), 254 (52), 237 (76) , 217 (27), 178 (30), 148
(53), 104 (92), 91 (50), 57 (55) and 43 (85) .
The structure of 4 was confirmed by acidic
hydrolysis as follows: The compound 4 (10 mg) was
suspended in HCI solution (0.5 N, 5 mL) and stirred
at room temperature for 30 minutes . The solution was
neutralized with 10% sodium bicarbonate solution
and extracted with ethyl acetate. The organic layer
was dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The hydrolyzed
product 5 gave positive ferric chloride test, which
indicates presence of hydroxyl group adjacent to
carbony I group. The structure of 5-hydroxy-3,3',4',5'-tetramethoxytlavone 5 was confirmed by
following physical and spectroscopic data. Rr:
0.18(CHCI 3); UV (CH 30H, AmaJnm): 347.8, 256.4 ;
IR (KBr/cn,-I): 3396.4,2922,2850, 1647, 1604, 1467,
1416, 1342, 1238, 1126.4 and 1012.6; IH NMR
(COCl 3): 8 3.84 (s, 3H, C r OCH 3), 3.91 (s, 9H, 3 x
OCH3), 7.07 (d, 1=7.4 Hz, IH, Cs-H), 7.43 (s, 2 H,
C 2'.6,-H ), 7.41 (d, 1=8.26 Hz, C 6 -H), 7.63 (t, I H, C r
1-1), 12.51 (s, I H, Cs-OH); J3 C NMR (OMSO-dr,) : 8
56.28 (C3'.5,-OCH 3 ), 60.07 (C 4 ,-OCH)), 60.94 (C)OCH 3), 106.03 (C 2 '.6' ), 116. 12 (C s), 117.25 (C IO ),
118.58 (C 6 ), 125.47 (CJ'), 133 . 10 (C 7), 140.48 (C 9 ) ,
141.55 (C 4'), 149.27 (CJ'.5'), 153 .10 (C 2), 154.40 (C s),
156.27 (C 3) and 173 .66 (C 4 ) ; EI-MS (mlz) : 358 (32),
342 (100), 327 (29), 312 (35), 298 (13), 222 (52), 178
(21), 162 (17),136 (53), 91 (51),57 (65).
Isolation of quercetin 6 from methanolic extract
of dry leaves of Quercus semicarpifolia. The organic
residue left after petroleum ether and acetone extract
226
INDIAN J. CHEM., SEC B, JANUARY 2004
of dried leaves of Quercus semicarpifolia was extracted with hot methanol for 56 hr using soxhlet. The
methanol solution obtained was concentrated under
reduced pressure. The residue obtained was absorbed
on silica gel and the compound was purified with column chromatography over silica gel (60-120 mesh).
Elution of column with chloroform: methanol (95: 5)
gave 15 mg of quercetin 6. R r: 0.37 (CHCI)); 308-10
°c (lit. 2 1 m.p. 311 °C); UV (CHCI), Am,.!nm): 258 .03,
375.43; IR (KBr/cm-' ): 3420, 1725, 1574, 1362 and
1113; 'H NMR (CDCl)): 8 12.31 (s, IH, C-5), 10.36
(s, IH, C-3), 7.6 (od, IH, J=6 .54 Hz, J=l.72 Hz, C6'),7.74 (d, IH, J=l.72 Hz, C-2'), 6.9 (d, IH, J=6 .54
Hz, C-5'), 6.37 (d, IH, J=1.8 Hz, C-8), 6.19 (d, IH,
J=1.8 Hz, C-6); EI-MS (rnIz): 302 (34), 221 (100),
193 (59), 148 (26), 119 (65),92 (40), 81 (28),55 (47)
and 44 (33) .
Acknowledgement
The authors are grateful to DBT, New Delhi for financial support. One of the authors (AK) is thankful
to the UGC, New Delhi for the award of SRF.
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