Phytochemical investigation of Tabebuia palmeri

Article abstract of DOI:10.1007/s10600-014-0818-y

Phytochemical investigation of the stems, flowers, and leaves of Tabebuia palmeri has led to the isolation of a novel compound, 6-(1-hydroxyundec-3-enyl)- tetrahydropyran-2-one, and 16 known compounds, viz. 1-hexadecanol, 1-triacontanol, stigmast-5-en-3β-ol, 2-hydroxy-3-(3-methylbut-2-enyl)-1,4- naphthoquinone (lapachol), methyl 3,4-dimethoxybenzoate, 2-acetyl-4H,9H- naphtho[2,3-b]furan-4,9-dione, 3,4-dimethoxybenzoic acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3β-hydroxy-12-ursen-28-oic acid, 5,7,4β-trihydroxyflavone, β-sitosteryl-β-D-galactoside, 6-O-(p-hydroxybenzoyl)-epiaucubin, 4-O-β-D-glucosylbenzoic acid, 9Z,12Z-octadecadienoic acid, and hexadecanoic acid. Except lapachol, all these phytoconstituents were isolated from the Tabebuia palmeri for the first time, and their structures were established on the basis of spectral data analysis and chemical reactions.

Full text of DOI:10.1007/s10600-014-0818-y

Chemistry of Natural Compounds, Vol. 49, No. 6, January, 2014 [Russian original No. 6, November–December, 2013]
PHYTOCHEMICAL INVESTIGATION OF Tabebuia palmeri
1
1
2
Rajeev Sakhuja, Monika Vashist, Yudhvir K. Bhoon,
UDC 547.918 + 547.972
1
*
and Subhash C. Jain
Phytochemical investigation of the stems, flowers, and leaves of Tabebuia palmeri has led to the isolation of
a novel compound, 6-(1-hydroxyundec-3-enyl)-tetrahydropyran-2-one, and 16 known compounds, viz.
1
(
-hexadecanol, 1-triacontanol, stigmast-5-en-3ꢀ-ol, 2-hydroxy-3-(3-methylbut-2-enyl)-1,4-naphthoquinone
lapachol), methyl 3,4-dimethoxybenzoate, 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione, 3,4-dimethoxybenzoic
acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3ꢀ-hydroxy-12-ursen-28-oic acid, 5,7,4ꢁ-trihydroxyflavone,
-sitosteryl-ꢀ-D-galactoside, 6-O-(p-hydroxybenzoyl)-epiaucubin, 4-O-ꢀ-D-glucosylbenzoic acid,
Z,12Z-octadecadienoic acid, and hexadecanoic acid. Except lapachol, all these phytoconstituents were
ꢀ
9
isolated from the Tabebuia palmeri for the first time, and their structures were established on the basis of
spectral data analysis and chemical reactions.
Keywords: Tabebuia palmeri, Bignoniaceae, 6-(1-hydroxyundec-3-enyl)-tetrahydropyran-2-one.
Tabebuia palmeri (Bignoniaceae) is a medium sized tree found mostly in tropical America. The bark decoction is
reported to be highly effective against cancer [1]. Plants belonging to the family Bignoniaceae, particularly to the genus
Tabebuia (Tecoma), are major sources of naphthoquinones possessing several biological and pharmacological activities [2–4].
Only one report has appeared in the literature earlier on the isolation of quinones and 4-aryltetralin derivatives [1] from the
bark of Tabebuia palmeri. This paper describes for the first time the isolation and structural elucidation of the phytoconstituents
of the flowers, stems, and leaves of Tabebuai palmeri.
The air-dried stems of Tabebuia palmeri were extracted by methanol, and the viscous dark brown mass was adsorbed
on silica gel (60–120 mesh) for the preparation of slurry. The slurry was chromatographed over a silica gel column, packed in
petroleum ether and eluted successively with mixtures of CHCl –petroleum ether, followed by mixtures of CHCl –MeOH,
3
3
yielding in all nine pure compounds 1–9.
The air-dried flowers of T. palmeri were extracted with methanol, and the pale yellow mass was adsorbed on silica gel
(
60–120 mesh) for the preparation of slurry. The slurry was chromatographed over a silica gel column, packed in CHCl and
3
eluted successively with mixtures of CHCl –MeOH, yielding nine pure compounds 3, 8, 9, and 10–15.
3
O
O
HO
O
OH
1
O
4
'
O
HOH
2
C
OH
OH
1
1'
O
5
2
'
5
3
O
OH
RO
3
, 13
10
14
OH
3: R = H; 13: R = Gal
1) Department of Chemistry, University of Delhi, 110007, Delhi, India, fax: 91 11 27666605, e-mail: jainsc48@hotmail.com;
2
) Department of Chemistry, Sri Venkateswara College, University of Delhi, 110021, Delhi, India. Published in Khimiya
Prirodnykh Soedinenii, No. 6, November–December, 2013, pp. 891–894. Original article submitted August 23, 2012.
0
0
009-3130/14/4906-1039 2014 Springer Science+Business Media New York
1039

1
13
TABLE 1. H (300 MHz) and C NMR (75 MHz) Data of Compound 10 (CDCl , ꢂ, ppm, J/Hz)
3
C atom
ꢂC
ꢂH
C atomꢄ
C
ꢂ ꢄ
H
ꢂ ꢄ
2
3
4
5
6
176.2
32.2
30.0
29.5
70.1
63.3
34.4
–
5ꢁ
6ꢁ
7ꢁ
8ꢁ
9ꢁ
31.9
28.4
25.6
25.2
23.9
23.0
14.4
2.03 (2H, m)
2.30 (2H, t, J = 7.3)
1
.60 (4H, m)
1.25 (10H, br.s)
4.26 (1H, dd, J = 6.2, 12.0)
4.16 (1H, dd, J = 4.3, 12.0)
2.78 (2H, m)
1
ꢁ
10ꢁ
11ꢁ
2ꢁ
0.88 (3H, t, J = 6.6)
3
4ꢁ
ꢁ
1
31.5, 129.3
5.36 (2H, m)
The air-dried leaves of T. palmeri were extracted with CH Cl –MeOH mixture, and the green mass was adsorbed on
2
2
silica gel (60–120 mesh) for the preparation of slurry. The slurry was chromatographed over a silica gel column, packed in
CHCl and eluted successively with mixtures of CHCl –MeOH, yielding three pure compounds 1, 16, and 17.
3
3
The physical and spectroscopic data of the known compounds isolated agreed with those reported in the literature for
naphthoquinones: lapachol (4) [5], 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione (6) [6]; flavonoid: 5,7,4ꢁ-trihydroxyflavone
(
(
12) [7]; steroids: stigmast-5-en-3ꢀ-ol (3) [8], ꢀ-sitosteryl-ꢀ-D-galactoside (13) [9]; aromatic acids: methyl 3,4-dimethoxybenzoate
5) [10], 3,4-dimethoxybenzoic acid (7) [11], 3,4-dihydroxybenzoic acid (8) [11], 4-hydroxybenzoic acid (9) [11],
4
-O-ꢀ-glucosylbenzoic acid (15) [12]; iridoid: 6-O-(p-hydroxybenzoyl)-epiaucubin (14) [13]; aliphatic compounds: 1-hexadecanol
(
1) [14], 1-triacontanol (2) [15], linoleic acid (16), and palmitic acid (17).
Compound 10 is a new natural product isolated from the methanol extract of flowers as a colorless oil. Its molecular
formula was determined to be C H O on the basis of its EI-MS (m/z 268), indicating three degrees of unsaturation. Its IR
1
6 28 3
–
1
spectrum showed a characteristic absorption band of a six-membered lactone at 1742 cm along with a peak of a hydroxyl
–
1
group at 3427 cm . The presence of these functional groups became more evident by the chemical shift value of lactone
carbonyl at ꢂ 176.2 and two other peaks at ꢂ 63.3, 70.1 in its 13C NMR spectrum. However, 10 did not form any acetate under
normal conditions (Ac O–pyridine), indicating that the hydroxyl group was either secondary or tertiary. Atwo-proton multiplet
2
1
13
at ꢂ 5.36 in its H NMR spectrum and their corresponding carbons at ꢂ 129.3 and 131.5 in its C NMR spectrum suggested the
presence of two olefinic protons in the molecule. A two-proton multiplet at ꢂ 2.03 and another two-proton triplet at ꢂ 2.30 were
assigned for the allylic methylene and methylene ꢃ to the carbonyl group. Two double doublets each integrating for one proton
at ꢂ 4.16, 4.26 (J = 12 Hz each) were assigned to two methines adjacent to oxygen and the carbon bearing the hydroxyl group
in the molecule. A two-proton multiplet at ꢂ 2.78 was assigned for the methylene in between the olefinic carbon and the carbon
bearing a hydroxyl group. The rest of the methylenes of the long chain and that of the lactone ring were observed at ꢂ 1.25 and
1.60 as ten and four protons, respectively. Two characteristic peaks at m/z 256 and 139 were observed in its EI-MS as a result of
fragmentation of ꢂ-lactones and allylic cleavage, respectively. On the basis of the above discussion, compound 10 was
characterized as 6-(1-hydroxyundec-3-enyl)-tetrahydropyran-2-one, which happened to be a novel natural product.
The insecticidal activity of compound 10 was determined against Bruchus chinensis (Coleoptera: Bruchiae) by studying
the oviposition inhibition assay following our earlier reported procedure [16]. Preliminary data suggested that compound 10
showed oviposition inhibition in B. chinensis. Thus, insects have susceptibility and oviposition deterrency to compound 10,
which could be used for the disruption of egg laying in the field and stored grain godowns to reduce the pest population.
However, since the compound was isolated in very low amounts, its detailed toxicology studies against B. chinensis and other
insects could not be carried out.
Compound 11 was characterized as 3ꢀ-hydroxy-12-ursen-28-oic acid, commonly known as ursolic acid on the basis
of spectral data and comparison of the observed melting point with that reported in the literature [17]. The assigned structure
of 11 was further confirmed by carrying out its esterification with diazomethane and acetylation with acetic anhydride in
1
pyridine. The H NMR spectra of the ester and acetate of 11 confirmed the formation of a methyl ester and monoacetate, thus
further supporting the assigned structure.
The presence of 11 in the flowers makes them more useful as 11 was known to show antioxidative [18, 19],
anti-inflammatory [20], and anticancer activity against several human cell lines [21–24], and also pro-apoptotic [25] activities.
1
040

EXPERIMENTAL
General Procedures. Melting points were determined on a sulfuric acid bath and are uncorrected. Column
chromatography was performed on silica gel (60–120 mesh, Merck), with gradient elution using petroleum ether, CHCl , and
3
CH OH in increasing polarity, and analytical thin layer chromatography (TLC) was performed using silica gel G coated TLC
3
1
13
plates (Merck). H (300 MHz), C (75 MHz) NMR spectra were recorded on a Bruker AM 300 spectrometer in CDCl3,
CD OD, and CD SOCD IR spectra were recorded on a Shimadzu model 435 spectrophotometers as thin film or KBr discs.
3
3
3.
Mass spectra were recorded on a varian MAT 311A instrument using electron ionization (EI) at 70 eV and TOF MS on LCT
micromass. For insecticidal studies, B. chinensis, a pulse beetle, was used to examine the activity of compound 10. For this
purpose, adult insects of B. chinensis were collected from the stored grain house, and their cultures were maintained in the
laboratory at 28 ꢅ 2ꢆC, 75 ꢅ 5% relative humidity, and a photoperiod of 12:12 (L:D) h. The insects were reared on gram seeds
at 10–12% moisture content.
Plant Material. Tabebuia palmeri was introduced by the Plant Introduction Division in National Botanical Research
Institute (NBRI), Lucknow, India in the late eighties and was brought from the NBRI Nursery. It was grown in Haryana in
2
002. The stems, flowers, and leaves were collected from the Gurgaon region located in Haryana, India in January 2007. The
plant was identified by Dr. S. C. Sharma, Head of the Department, Garden Division, NBRI, Lucknow, India.
Extraction and Isolation. Dried stems (700 g) were chopped and extracted with MeOH (4 L) for 24 h in a Soxhlet
apparatus. Dried flowers (24 g) were crushed and extracted with MeOH (1 L) for 24 h at room temperature. Dried and milled
leaves (1200 g) were extracted with CH Cl –MeOH (1:1) (2 L) for 24 h at room temperature. Liquid extracts were evaporated
2
2
under reduced pressure to dryness. The resulting extracts of stems (40 g), flowers (4.6 g), and leaves (36 g) were chromatographed
over a silica gel column separately, and 17 compounds were isolated by different purification techniques.
Methanol Extract of the Stems. The MeOH extract (40 g) of the stems was packed over a silica gel column
chromatograph in petroleum ether. Elutions with chloroform–petroleum ether (10:90, 20:80, 50:50) followed by CHCl and
3
then MeOH–CHCl (2:98, 5:95, 10:95, 20:80) and MeOH provided a total of 178 fractions (400 mL each), of which similar
3
composition fractions as determined by TLC were pooled to get 12 major fractions. These 12 fractions were subjected to
repeated silica gel column chromatography, preparative thin-layer chromatography, and crystallization techniques to yield
nine compounds in pure form. The above techniques afforded hexadecanol (1, fraction 1–15, 86 mg, R 0.6; petroleum ether–EtOAc
f
(
98:2)), triacontanol (2, fraction 6–31, 8 mg, R 0.4; petroleum ether–EtOAc (98:2)), stigmast-5-en-3ꢀ-ol (3, fraction 32–39,
f
1
8 mg, R 0.3; petroleum ether–EtOAc (97:3)), lapachol (4, fraction 55–65, 990 mg, R 0.5; petroleum ether–chloroform
f f
(
1:1)), methyl 3,4-dimethoxybenzoate (5, fraction 66–74, 60 mg, R 0.7; CHCl ), 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-
f 3
dione (6, fraction 75–87, 5 mg, R 0.2; MeOH–CHCl (2:98)), and 3,4-dimethoxybenzoic acid (7, fraction 75–87, 36 mg,
f
3
R 0.2; MeOH–CHCl (5:95)), 3,4-dihydroxybenzoic acid (8, fraction 107–126, 210 mg, R 0.2; MeOH–CHCl (12:88)), and
f
3
f
3
4
-hydroxybenzoic acid (9, fraction 88–106, 258 mg, R 0.2; MeOH–CHCl (10:90)).
f 3
Methanol Extract of the Flowers. The MeOH extract (4.6 g) of the flowers was chromatographed using silica gel
column chromatography in CHCl , followed by elutions with MeOH–CHCl (2:98, 5:95, 10:90, 20:80, 50:50) and finally with
3
3
1
00% MeOH of 120 mL each to collectively get 89 fractions. Similar fractions were mixed as per their TLC behavior to yield
eight major fractions. From these fractions nine compounds were isolated in pure form, of which stigmast-5-en-3ꢀ-ol (3),
,4-dihydroxybenzoic acid (8), and 4-hydroxybenzoic acid (9) were also isolated from the stems. The remaining six compounds
were characterized as 6-(1-hydroxyundec-3-enyl)-tetrahydropyran-2-one (10, 3 mg, fraction 1–8, R 0.4, petroleum ether)
3
f
3
ꢀ-hydroxy-12-ursen-28-oic acid (11, fraction 9–19, 37 mg, R 0.4, EtOAc–petroleum ether (30:70)), 5,7,4ꢁ-trihydroxyflavone
f
(
12, fraction 20–27, 4 mg, R 0.3, MeOH–CHCl (8:92)), ꢀ-sitosteryl-ꢀ-D-galactoside (13, fraction 28–32, 5 mg, R 0.6,
f
3
f
MeOH–CHCl (12:83)), 6-O-(p-hydroxybenzoyl)-epiaucubin (14, fraction 33–38, 400 mg, R 0.2, MeOH–CHCl (15:85)),
3
f
3
and 4-O-glucosylbenzoic acid (15, fraction 68–89, 20 mg, R 0.5, MeOH–CHCl (20:80)).
f
3
–
1
6
-(1-Hydroxyundec-3-enyl)-tetrahydropyran-2-one (10). Colorless oil. IR (KBr, ꢇ , cm ): 3427, 2920, 2852,
max
1
3
1
742, 1463, 1377, 1164, 1377, 1164, 1099, 721. For PMR and C NMR spectral data, see Table 1. Mass spectrum (EI, 70 eV)
m/z, Irel., %): 268 (M , 93), 256 (22), 244 (12), 238 (58), 224 (25), 210 (31), 194 (37), 181 (37), 168 (48), 153 (58), 139 (80),
+
(
+
1
25 (100). HR-TOF-MS m/z 269.2122 [M + H] (calcd for C H O , 269.2107).
1
6 29 3
3
ꢀ-Hydroxy-12-ursen-28-oic Acid (11). Colorless needles, mp 288–290ꢆC (lit. [17] mp 289–292ꢆC). It gave a positive
Liebermann–Burchard test for triterpenes. It also gave a pale yellow color with tetranitromethane.
Esterification of 11. Compound 11 (10 mg) was subjected to esterification with excess diazomethane. The solvent
was evaporated to get a pure esterified product. PMR spectrum (300 MHz, CDCl , ꢂ, ppm, J/Hz, 0 = TMS): 5.24 (1H, m, H-12),
3
1
041

3
.60 (3H, s, COOCH ), 3.20 (1H, m, H-3ꢃ), 2.22 (1H, d, J = 11.0, H-18), 1.99–1.21 (m, cyclic methylenes and methines), 1.07
3
(
3H, s), 0.98 (3H, s), 0.94 (3H, s), 0.92 (3H, d, J = 5.9), 0.85 (3H, d, J = 6.0), 0.78 (3H, s), 0.74 (3H, s).
Acetylation of 11. Compound 11 (15 mg) was refluxed with acetic anhydride (0.5 mL) and dry pyridine (0.2 mL) for
about 2 h. The contents were kept overnight at room temperature. Crushed ice was subsequently added to it, and the white
precipitate that separated was filtered, washed with water, and dried. It was recrystallized from petroleum ether–chloroform to
give pure acetate (mp 190–192ꢆC, R 0.43 in CHCl as developing solvent). PMR spectrum (300 MHz, CDCl , ꢂ, ppm, J/Hz,
f
3
3
0
= TMS): 5.25 (1H, m, H-12), 4.99 (1H, m, H-3ꢃ), 2.18 (1H, d, J = 1.2, H-18), 2.04 (3H, s, OCOCH ), 1.98–1.29 (m, cyclic
3
methylenes and methines), 1.08 (3H, s), 0.95 (6H, s), 0.85 (9H, br.s), 0.79 (3H, s).
Dichloromethane/Methanol Extract of the Leaves. The CH Cl –MeOH extract (36 g) was subjected to silica gel
2
2
column chromatography in chloroform. Elutions with increasing polarities with MeOH–CHCl (2:98, 5:95, 10:90, 20:80) led
3
to the collection of 92 fractions. These fractions were separately screened on TLC to reduce the number of fractions to 10
fractions. However, most of the above fractions contained major amounts of chlorophyll and other green components in them.
Thus, only 3 long-chain aliphatic compounds were isolated, viz. 9Z,12Z-octadecadienoic acid (16, 20 mg, R 0.2, EtOAc–CHCl
f
3
(
3:97)), hexadecanol (1, 36 mg R 0.7, petroleum ether), and hexadecanoic acid (17, 40 mg, R 0.1, CHCl ).
f
f
3
ACKNOWLEDGMENT
The authors are thankful to the Council of Scientific and Industrial Research (CSIR, India) and the University Grant
Commission (UGC) for financial assistance.
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042