BlOTROPIA No


BlOTROPIA No. 11, 1998: 1 - 8 

STUDIES ON NATURAL PRODUCTS OF ALBIZIA SP. 

HILMAN AFFANDI, ARIFNURYADIN 

SEAMEO BIOTROP, P.O. Box 116, Bogor 16001, Indonesia 

and 

ROGER W. READ 
Department of Organic Chemistry, The New South Wales University, 

 P.O. Box 1 Kensington, N.S.W. 2033, Australia 

ABSTRACT 

The bark of Albizia lebeckioides and Albizia falcataria have been examined for their chemical 
constituents. A. lebeckioides has yielded the steroidal ketone, stigmasta-4,22-dien-3-one, and a triterpene alcohol 
as the major neutral components. A. falcataria has yielded a similar triterpene and fatty ester as its major 
constituents. The toxicity of the compounds was evaluated in bioassay against termites. 

The results showed that lupenone and stigmastadienone were toxic ioNeotermes dalbergiae species. In 
contrast these compounds were less toxic to Cryptotermes cynocephalus species. 

Key words: Insecticidal plants / Albizia lebeckioides I Albizia falcataria I Bark / Extracts / Chemical 
constituents / Toxicity / Termites 

INTRODUCTION 

Some substances are produced by plants reasons for such as defence, growth 
regulation, hormonal action or as waste products. In teak (Tectona grandis L.f), for 
example, tectoquinone is produced to protect these trees against termite attack 
(Romanis 1887; Oshima 1919; Wolcott 1946). Resin ofPinus possesses a number of 
biologically active natural products. A major constituent, a-pinene is well known for its 
antimicrobial and insecticidal properties (Bridges 1987). 

Among fast growing trees, Albizia falcataria was reported to resist termite 
attack (Kalshoven 1950; Griffioen 1954). Laboratory tests designed to study the 
resistance of Albizia falcataria against dry wood termites (Cryptotermes 
cynocephalus) revealed that the resistance of Albizia falcataria lies somewhere 
between that of teak and Hevea brasiliensis or even closer to teak (Tarumingkeng and 
Martawidjaja 1969). Another species of Albizia e.g. Albizia lebeckioides was reported earlier 
to contain toxic alkaloids (Greshoff 1914). 

These reports suggested that both species of Albizia may contain useful 
chemicals that resist termite attack, therefore a search for biologically active 

1 
 



BIOTROPIA No. 11, 1998 

compounds from the bark of Albizia falcataria and Albizia lebeckioides was 
undertaken. The objective of this study was to isolate and characterize the main 
chemical components and to determine their toxicity to termites. 

MATERIALS AND METHODS 

General 

'H NMR spectra were recorded at 500 MHz and 13C NMR spectra were 
measured at 125.6 MHz on a Bruker AM 500 spectrometer. Mass spectra were 
recorded at 70 eV with an A.E.I. MS 12 spectrometer. All measurements were 
performed at the School of Chemistry, the University of New South Wales, Australia. 

Initial column chromatographic separations were made on Merck 60 silica gel. 
Merck Kieselgel 60 F254 was employed for preparative TLC with a 1 mm layer of 
adsorbent on 20 x 20 cm glass plates and 200 mg of material applied to each plate. 
Analytical TLC was performed on commercial aluminium-backed Merck Kieselgel 60 
F254 Art. 5554 plates, visualizing under UV 254 nm light or charring after applying a 4% 
H2SO4/MeOH spray. 

Plant Material 

Samples of bark of Albizia falcataria were collected from Kesatuan Pemangkuan 
Hutan (KPH), Kediri while the bark of A. lebeckioides was taken from Batutulis, Bogor. 

Extraction and Isolation 

Dried powdered bark of Albizia lebeckioides (1.50 kg) was extracted with 
methanol to give crude extract (78.15 g). The crude extract was then re-extracted with ether 
to generate an ether soluble fraction (27.77 g) and an insoluble residue (50.37 g). 
The ether soluble fractions were carefully fractionated by column chromatography 
(silica gel 60) eluting with hexane containing increasing amounts of ethyl acetate to give 6 
fractions. Fraction 4 (1.12 g) and fraction 5 (0.98 g), as two major fractions, were 
subjected to purification by preparative thin-layer chromatography (ethyl acetate - 
hexane, 1 : 9). Fraction 4 gave a solid material 

 

2 
 
 



Studies on Natural Products ofAlbizia sp.- Hilman Affandi et al. 

(0.85g) which crystallized from chloroform to give stigmasta-4,22-dien-3-one (2) (0.67 
g) m.p. 181-184°. Fraction 5 generated a crystalline solid (0.76 g). Recrystallization of 
the latter gave lupeol (1) (0.51 g) m.p. 198-199°. 

The bark of A. falcataria (1.50 kg) was treated in a similar manner to the 
method above. Re-extraction of the crude extract (65.83 g) with ether gave an ether 
soluble fraction (16.20 g) and a residue (49.62 g). Fractionation of the ether soluble 
portion by column chromatography eluting with hexane generated 6 fractions. 
Purification of the fastest moving fraction (1.28 g) by preparative thin-layer 
chromatography (ethyl acetate-hexane, 1:9) gave a solid material (0.93 g) which 
crystallized from chlorform to give fatty ester (4) (0.82 g). Fraction 2 (1.04 g), upon thin 
layer chromatography (ethyl acetate-hexane, 1:9) gave a crystalline solid (0.78 g). 
Recrystallization of the solid from chloroform gave lupenone (3) (0.68 g) m.p. 160-161°. 

Termite Bioassays 

The toxicity of the pure constituents was evaluated by bioassay using the termite 
Neotermes dalbergiae, obtained from commercial sources in Bogor, and Cryptotermes 
cynocephalus, collected from the Forest Product Research Institute, Bogor. 

Pure compounds (25 mg) were diluted with dichloromethane (0.5 ml). The solutions 
were applied to 4.5 cm Whatman no. 1 filter paper dish. After the solvent had been 
evaporated at ambient temperature, the filter papers were placed in 5 cm glass petri dishes, 
and 25 termites added to each container. Control chambers were treated in a similar manner 
using pure chloroform in place of solutions of the test subtances. Each pure compound was 
tested in four replicate experiments. 

At daily intervals the number of termites surviving was recorded, dead termites were 
removed, and the filter papers were moistened with distilled water. Dishes were kept 
covered in a darkened room at ambient temperature. 

RESULTS AND DISCUSSION 

Structural Analysis 

Methanolic extracts of the bark of A. lebeckioides and A. falcataria were re-
extracted with ether. The ether soluble extracts were carefully fractionated by column 
chromatography (silica gel) and purificed by preparative thin-layer chromatography 

 

 

 

3 



 
BIOTROPIA No. 11, 1998 

to yield lupeol (1) and stigmata-4, 22-dien-3-one (2) from die bark of A. lebeckioides, and 
lupenone (3) and fatty ester (4) from the bark ofA.falcataria. 

The structure of compound (1) was established from its microanalytical and 
spectroscopic data. Elemental analysis and mass spectrometry indicated the molecular 
formula C30H50O. The 'H n.m.r. data showed six singlet resonances at 5 0.82, 0.89, 0.93, 
0.98, 1.02 and 1.07, arising from six methyl groups attached to quaternary carbons. 
There also appeared one isolated singlet at 5 1.68 due to a vinylogous methyl group, 
and since the olefmic proton signals at 8 4.56 (dd, J 2.2, 1 4 Hz) and 4.68 (d J 2.2 Hz) were 
consistent with a 1,1-disubstituted alkene, a propenyl group was likely. Low field signals 
resonated at 5 3.18 (dd, J 10,.9, 5.3 Hz), 2.38 (dt, J 5.7, 11.1 Hz) and 1.91 (m), but there 
were no other olefmic resonances. The n.m.r. signal at 8 3.18 was suggestive of an axial 
proton adjacent to a quarternary centre and attached to a carbon bearing an hydroxyl group. 
The I3C n.m.r. spectrum (Table 1) -supported the structural assignment of (1). In particular 
there appeared seven methyl signals, only two olefmic carbon signals (8 109.3 (CH2) 
and 151.0 (C), and a low field methine resonance (8 78.9) for C3. The identity of 
compound (1) as lupeol was confirmed by melting point and mixed melting point with an 
authentic sample. 

Lupeol has been found in many plant extracts, including the resin of Pistacia 
leuticus (Marner et al. 1991). 

Compound (3) was obviously closely related to lupeol (1). Its mass spectrum gave 
a molecular ion at m/z 424, corresponding to two fewer hydrogens than lupeol. The 'H 
n.m.r. spectrum (Table 2) of compound (3) resembled that of (1), including the 
appearance of coupled olefmic signals at 8 4.57 and 4.70, and a deshielded methyl signal 
due to H3 in lupeol (1) was absent. Instead, there appeared additional signals at 8 2.4, 
corresponding to protons adjacent to a multiple bond. The compound was thereby 
identified as lupenone (3) and the structure was confirmed by mixed melting point with an 
authentic sample. 

Compound (2) differed from compounds (1) and (3). Elemental analysis and mass 
spectrometry indicated through its molecular formula, C29H46O, that it was a nor-
triterpene. There appeared in its 'H n.m.r. spectrum two methyl singlets, 8 0.57, 1.01, 
three methyl doublets, 8 0.79, 0.84, 1.03, and a methyl triplet, 8 0.80. Also there appeared a 
three proton multiplet corresponding to two internally coupled, trans olefmic protons 
(8 5 03, dd, J 15.1, 8.4 Hz, and 5 15 (dd, J 15.1, 8.4 Hz) and an isolated olefmic proton 
(8 5.18, m) with long range coupling, but no other signals at higher chemical shift than 
2.5 ppm. The presence of a 1,2-disubstituted and a 1,1,2-trisubstituted olefin was 
supported by the presence of signals at 5 117.0 (CH), 129.6 

 
 
4 

Studies on Natural Products ofAlbizia sp.- Hitman Affandi el al. 



(CH), 138.1 (CH) and 139.5 (C) in the 13C n.m.r. spectrum. The presence of a 
quaternary signal at 8 212.0 also confirmed the occurrence of a carbonyl group in the 
compound. Structure (2) was assigned from these data and was confirmed by mass 
spectrometric fragments at m/z 367 (M-C3H7), 298 (M-C8Hi6), 271 (M-CioH,9), 269 (M-
Ci0H2i), and comparative melting point. 

 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.   Structures of chemical constituents isolated fro

falcataria (3) 

Table 1.     13C n.m.r. chemical shift data of compounds (l)-(
 

C 1 
1 
2 
3 
4 
5 
6 
7 
8 
9 

10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 

39.3 
34.3 
55.3 
47.0 
59.3 
19.4 
33.0 
39.9 
50.5 
37.1 
21.8 
26.4 
39.7 
41.2 
26.5 
30.1 
44.7 
37.6 
52.5 

150.6 
30.2 
23.5 

 
 

2. STIGMASTA - 4,22-DIEN-3
3. LUPENONE
m the bark of A lebeckioides (1 and 2) and A. 

3) (ppm in CDClj) 

2 3 
37.3 
31.6 

212.2 
71.8 
42.3 

121.1 
33.9 
31.7 
50.2 
36.5 
20.9 
38.6 
42.2 
56.6 
24.1 
28.2 
56.1 
11.6 
19.4 
40.8 
21.5 

138.8 

39.7 
34.1 

218.4 
47.3 
54.9 
19.6 
33.0 
40.3 
50.6 
36.9 
21.9 
26.7 
39.5 
41.0 
26.4 
30.1 
44.9 
37.7 
52.5 

150.8 
30.3 
23.2 

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BIOTROPIA NO. 11. 1998 
 

Table 1. Continued 
 

C 1 2 3 
23 
24 
25 
26 
27 
28 
29 
30 

26.8 
20.9 
16.2 
15.4 
14.3 

151.0 
101.0 

20.6 

129.5 
51.4 
32.2 
19.2 
21.3 
25.7 
12.5 

- 

26.8 
20.9 
16.3 
15.4 
14.2 

151.1 
101.8 

20.7 

 
Table 2. 1H n. m.r spectral data of compound 1-3 (ppm in CDC13) 

 
H 1 2 3 

H-2 
H-3 
H-6 

H-18 
H-19 
H-22 
H-23 
H-24 
H-25 
H-26 
H-27 
H-28 
H-29 

2.37 – 2.55 m 
3.85 m 

- 
- 

1.2 – 2.0 m 
- 

1.07 s 
1.02 s 
0.93 s 
0.98 s 
0.89 s 

- 
4.56 - dd, 4.68 d 

- 
- 

5.45 d 
0.57s 
1.01 1 

5.15 dd 
5.03 dd 

- 
- 

0.79 d 
0.84 d 

-1.50m 
0.80 1 

2.36 - 2.60 m 
- 
- 
- 

1.5 -2.0m 
- 

1.07s 
1.19s 
0.94s 
0.97s 
0.89s 

- 
4.57 dd, 4.70 d 

 
Compound (4) could not be obtained pure and its structure could not be fully 

elucidated. From its 'H n.m.r. spectrum the substance was not terpene derived but was 
probably a fatty ester. There appeared in the low field region of the spectrum two triplets of 
equal intensity corresponding to methylene protons adjacent to oxygen (8 4.05) and a 
carbonyl group (8 2.28), respectively. In addition, there was a triplet resonance at 8 
0.88 that could be assigned to coincident signals from the methyl groups at the ends of 
aliphatic chains. The remaining signals comprised two narrow multiplets at 8 1.25 and 8 
1.57 corresponding to groups of similar methylene protons. The mass spectrum gave 
significant ions at m/z 425 and 205 that could not be assigned to any sensible structure 
or fragments. 

Toxicity of Compounds (l)-(3) 

A laboratory bioassay was conducted to assess the toxicity of pure compounds (l)-
(3), the tentatively assigned fatty ester (4), and the remaining extracts, to termites. The 
results showed that 53%, 48%, 42%, 54%, and 64%, respectively, of Neotermes 

 

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Studies on Natural Products ofAlbizia sp.- Hitman Aff'andi et al. 

dalbergiae termites that were fed these substances remained alive after the fourth day of the 
experiment (Diagram 1). 

While termites in control groups moved actively when the dish was uncovered for 
counting, temites in treatment groups did not. This observation may suggest that termites 
receiving chemical treatment responded in a general rather than specific manner to pure 
compounds. 

Specific responses such as death caused by direct application onto the termites were 
not observed. In addition the total termite mortality was not as high as expected from related 
studies using Cryptotermes cynocephalus termites (Diagram 2). This may have been due to 
insufficient doses of the pure compounds rather than low activity. 

Comparison of the relative toxicity of the compounds to Neotermes dalbergiae showed 
that lupenone and stigmastadienone were the most toxic compounds (Diagram I). 

In contrast lupenone and stigmastadienone were less toxic against the Cryptotermes 
cynocephalus species. This might be due to the fact that Neotermes dalbergiae, a drywood 
termite, does not attack living trees as does the Cryptotermes cynocephalus. Therefore, the 
former are more susceptible to offensive chemicals, because they have not developed 
resistance mechanisms (Messer 1990). 

 
 
 
 

 
 
 
 
 
 
 
 
 
 
 

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 Diagram 1. Toxicity of the compounds against Neotermes dalbergiae 
 
 
 
 
 
 
 
 
 

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BIOTROPIA No. 11, 1998 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

%
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Diagram 2    Toxicity of the compounds againts Cryptotermes cynocephalus 

CONCLUSION 

This study has shown that lupenone and stigmastadicnone, contained in the bark of 
Albizia falcataria and Albizia lebeckioides respectively, have biological activity against 
the drywood termite Neotermes dalbergiae and that in function they may play a defensive 
role. Toxic alkaloids, previously reported to be present, (Heyne 1984) have not been 
found in the bark of Albizia falcataria. 

REFERENCES 

APSIMON, J. 1973. The Total Synthesis of Natural Products, Vol 2, John Wiley & Sons, London. 
BRIDGES, J.R. 1987. Effect of Terpenoid Compounds on Growth of Symbiotic Fungi Associated with 

Southern Pine Beetle. Phytopathology, p. 77. 
FffiSER, L.F. AND M. FffiSER. 1959. Steroids, Van Nostrand Reinhold Company, London. 

GRIFFIOEN, K. 1964. Albizia falcataria sen goede industrie houtsoort. Tectona 63: 97-100. 
GRESHOFF, M. 1914. Indische Vergiftrapporten, No. 3 in Heyne, K. 1987. Tumbuhan Berguna Indonesia, 

Ruygrok & Co., Jakarta, p. 1021-1040. 
KALSHOVEN, L.C.B. 1950. De plagen van de cultuurgewassen in Indonesia. Deel I: 145-150. 
MARKER, F.J., FREYER, A., LEX, J. 1991. Interpenoid from Gum Mastic, The Resin of Pistacia leuticiis. 

Phytochemistry, 30 (11). 
MESSER, A.C. 1990. Chemical Ecology in an Indonesian Context, Ph.D. Thesis, Cornell University. OSHIMA, M. 

1919. Formesan Termites and Method of Preventing their Damage. Philip. Sul. 15: 319-386. ROMANIS, R. 1887. 

Certain Products from Teak. Chem. Soc. 54: 868. 
TARUMINGKENG, R.C. AND A. MARTAWIDJAJA. 1969. Pengujian Kayu Djeundjing (Albizia falcataria Backer) 

Terhadap Rayap Kayu Kering (Cryptotermes cynocephalus Light) Secara Laboratories. Rimba 
Indonesia, XIV (1-2) 

WOLCOTT, G.N. 1946. What to do about Pollila? Univ. Puerto Rico, Agric. Exp. Sta., Bull. No. 68. 
 
 
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