Unsaponifiable Lipid Constituents of Some Underutilized Tropical Seed Oils
Unsaponifiable Lipid Constituents of Some Underutilized Tropical Seed Oils
Unsaponifiable Lipid Constituents of Some Underutilized Tropical Seed Oils
Sterols, triterpene alcohols, and hydrocarbons present in the unsaponifiable fraction of some
underutilized tropical seed oils have been examined.The seeds include Telfairia occidentalis (TLO),
Andenopus breviflorus (ADB), Cucumeropsis edulis (CME), Antiaris africana (ATF), and Monodora
tenuifolia (MNT). The oil content of the seeds was high (34.7-68.8%), whereas triacylglycerols
comprised the dominant lipid group in the oils (65.4-73.9%). The percentage of unsaponifiables
ranged from 1.1 to 7.9%. Ten sterols were identified in the fractions. In the Cucurbitaceae oils (TLO,
CME, and ADB), ∆7-sterols constituted the dominant sterols. These include 24-ethylcholesta-7,-
22E,25-trienol (7), 24-ethylcholesta-7,25-dienol (9), 24Z-ethylidenecholes-7-enol (10), and 24-
ethylcholesta-7,24-dienol (11). However ∆5-sterols (1-5) occurred at the highest concentration in
the other two samples (ATF and MNT). Fifteeen triterpene alcohols were detected in the fractions.
Olean-12-enol (16), isomultiflorenol (8), and lupeol (23) were the dominant alcohols in the
Cucurbitaceae family, whereas R-amyrin (urs-12-enol) (20) was the dominant triterpene alcohol in
ATF and MNT. A mixture of C18-C34 n-alkanes, squalene, and some monoterpenes was detected in
the hydrocarbon fraction.
INTRODUCTION The high fixed oil content and the edibility charac-
teristics exhibited by some lesser known and underuti-
Unsaponifiable lipid constituents of seed oils contain lized seed oils have been studied by Esuoso and co-
a variety of bioactive substances, which includes hy- workers (Esuoso and Odetokun, 1995; Esuoso, 1996;
drocarbons, tocopherols, sterols, and terpene alcohols. Esuoso et al., 1998; Esuoso and Bayer, 1998). The
Terpene alcohols have shown both cytostatic and cyto- results from these studies have prompted us to examine
toxic properties (Jalad et al., 1977; Kupchan et al., the hydrocarbons and other weakly polar unsaponifi-
1978). These compounds are also known to possess ables in the oils. In addition, the roles of these groups
herbicidal and antimicrobial properties (Nakatani et al., of compounds on the biosynthesis of major compounds
1981; Dreyer and Trousdale, 1978). A large variety of in the oils are not well understood, and it is not yet clear
sterols have also shown insecticidal properties (Heft- whether the compounds could serve as chemotaxonomic
mann, 1970). To date, hydrocarbons represent the least markers for plant families. These factors coupled with
investigated fraction of the unsaponifiable matter of the the need to develop environmentally friendly natural
fixed oils. There have been limited studies on the nature insecticides are important considerations that stimu-
of hydrocarbons in vegetable oils. Early studies by lated our interest in examining the nature of these
Capela et al. (1963) revealed the presence of three unsaponifiables.
groups of hydrocarbons in vegetable oils. The authors This paper reports, for the first time, the unsaponi-
identified n-paraffins, homologues of C10-C35, and also fiables of five lesser known and underutilized tropical
made a presumptive identification of alkenes based on seed oils: Telfairia occidentalis (TLO); Andenopus
their results of iodine values. Recent studies on the breviflorus (ADB); Cucumeropsis edulis (CME); Antiaris
distribution and occurrence of sterols, triterpene alco- africana (ATF); and Monodora tenuifolia (MNT).
hols, and other unsaponifiable constituents of vegetable
oils have been reported (Andriamanantena et al., 1983; MATERIALS AND METHODS
Akihisa et al., 1986, 1988). Although many other studies
have been published on the analysis of unsaponifiables Collection and Pretreatment of Samples. The samples
of vegetable oils, much remains obscure and needs were obtained from selected markets in Ibadan, Warri, Akure,
further investigation. and Benin, all in southern Nigeria. They were identified at
the International Institute of Tropical Agriculture (IITA),
Ibadan, Nigeria, and thereafter stored for 3-4 weeks in plastic
* Corresponding author and a visiting Research Scientist bags at 4 °C prior to extraction and analysis.
from the Department of Chemistry, University of Ibadan, Extraction of the Oils. Seed samples were cleaned with
Ibadan, Nigeria (fax 49 7071 295 034; e-mail kayode.esuoso@ water and air-dried in the laboratory. The seeds were ground
uni-tuebingen.de). with a Christy laboratory mill (Comitrol processor model 3600).
† University of Ibadan. The powdered samples were extracted using a Soxhlet extrac-
‡ University of Tuebingen. tor with petroleum ether (40-60 °C) for 24 h. The extracted
Table 1. Systematic Names and Codes of the Five Table 2. Percentage Oil Content, Unsaponifiables, and
Tropical Seeds Studied Lipid Classes of Five Underutilized Tropical Seed Oilsa
code systematic name family plant % weight
TLO Telfairia occidentalis Cucurbitaceae species oilb UNSP HC TG FFA DG ST MG PL
CME Cucumeropsis edulis Cucurbitaceae TLO 48.6 1.1 1.3 70.0 0.2 2.7 2.0 1.6 3.6
ADB Andenopus breviflorus Cucurbitaceae CME 46.1 1.2 1.1 73.9 2.7 1.8 2.6 0.7 1.0
ATF Antiaris africana Moraceae ADB 55.1 1.3 1.0 70.1 1.4 1.1 1.6 0.5 0.6
MNT Monodora tenuifolia Annonaceae ATF 68.8 6.5 4.1 65.4 0.8 4.5 2.5 1.1 2.0
MNT 34.7 7.9 3.4 71.2 2.4 1.7 1.6 0.5 0.4
oil was dried over anhydrous sodium sulfate and the solvent a HC, hydrocarbons; TG, triacylglycerols; FFA, free fatty acids;
removed under reduced pressure in a rotary evaporator. DG, diacylglycerols; MG, monoacylglycerols; PL, polar lipids;
Lipid Separation. Lipids were separated into classes on UNSP, unsaponifiables. b Based on the percent dry weight of the
0.55 mm silica gel plates (20 × 20 cm). The mobile phase used seed.
was a solvent mixture of petroleum ether/diethyl ether/acetic
acid (80:20:1). It was developed according to the method Table 3. Relative Retention Times of Sterols and
described earlier (Esuoso et al., 1998). Triterpene Alcohols Used as References in Gas
Isolation of the Unsaponifiables. Oil (10 g) dissolved in Chromatography
200 mL of ethanolic potassium hydroxide (2 M) was refluxed code RRT compound
for 1 h. The reaction mixture was diluted to 400 mL with
distilled water and transferred to a separating funnel. The sterols
unsaponifiables were extracted three times with 100 mL of 1 1.00 cholesterol (cholest-5-enol)
2 1.17 24-methylcholesta-5,22E-dienol
diethyl ether. The ether extracts was first washed with 100 3 1.34 campesterol (24-methylcholesterol)
mL of aqueous solution of potassium hydroxide (0.5 M) to 4 1.46 stigmasterol (24-ethylcholesta-5,22E-dienol)
remove any residual free fatty acids. Further washing and 5 1.47 24-ethylcholesta-5,22E,25-trienol
cleaning was carried out five times with 100 mL of distilled 6 1.66 β-sitosterol (24-ethylcholesterol)
water, and the ether layer was dried over anhydrous sodium 7 1.68 24-ethylcholesta-7,22E,25-trienol
sulfate. The solution was filtered and the solvent removed in 8 1.84 isofucosterol (24Z-ethylidenecholesterol)
a rotary evaporator. The unsaponifiables fraction in the oil was 9 1.95 24-ethylcholesta-7,25-dienol
then expressed in weight percent. 10 2.17 avenasterol (24Z-ethylidenecholest-7-enol)
11 2.19 peposterol (24-ethylcholesta-7,24-dienol)
Separation of the Unsaponifiables. A chloroform solu-
terpene alcohols
tion (50%) of the unsaponifiable material (30 mg/plate) was 12 1.30 euphol (eupha-8,28-dienol)
then applied uniformly along a line from the edge of a 20 × 13 1.33 24-dihydrolanosterol (24-lanoest-8-enol)
20 cm plate coated with a 0.55 mm layer of silica gel and 14 1.50 tirucallol (tirucalla-8,24-dienol)
developed three times with hexane/ethyl acetate (6:1 v/v) as 15 1.58 taraxerol (D-friedoolean-14-enol)
mobile phase. After development, the plates were sprayed with 16 1.66 β-amyrin (olean-12-enol)
a solution of Rhodamine-6G in ethanol (0.5%) and observed 17 1.72 butyrospermol (eupha-7,24-dienol)
under UV light. Three different zones were marked: Rf 0.9- 18 1.77 isomultiflorenol (D:C-friedoolean-8-enol)
1.0, hydrocarbons (n-alkanes); Rf 0.4-0.5, triterpene alcohols; 19 1.79 24-methylenelanost-8-enol
20 1.87 R-amyrin (urs-12-enol)
and Rf 0.02-0.04, sterols. Each zone was carefully scraped
21 1.89 24-methylene-24-dihydroparkeol
from the plates and extracted thoroughly with diethyl ether. 22 1.90 cycloartenol (9β-; 19-cycloartenol)
Sterols and triterpene alcohols were silanated with 10 µL of 23 1.96 lupeol
bis(trimethylsilane)-trifluoroacetamide (BSTFA) at 60 °C for 24 2.10 24-methylenecycloartenol
1 h. The residue obtained was mixed with water and extracted 25 2.40 taraxasterol
with diethyl ether, and the solvent was removed at reduced 26 2.52 ι-taraxasterol
pressure in a rotary film evaporator.
Analysis of Unsaponifiables. Gas chromatograph Table 4. Composition of the Sterol Fraction of Five
Chrompack CP 9001 equipped with a flame ionization detector Underutilized Tropical Seed Oils
and a mosaic integrator was used for the studies. For the plant speciesa
determination of hydrocarbons, the fraction was injected into
the GC without derivatization using a capillary column (SE- RRT TLO ADB CME ATF MNT
54, 20 m × 0.27 mm, J&W Scientific, Köln, Germany). The 1.17 (2) - - - 2.2 4.2
programming was as follows: 35 °C for 3 min, temperature 1.34 (3) 3.8 5.9 1.2 3.2 5.8
increased at 5 °C/min to 280 °C, and held at 280 °C for 5 min. 1.46 (4) 2.6 3.8 4.2 13.8 12.6
Further determination was carried out on a GC-MS Varian 1.47 (5) 9.7 8.5 10.2 38.2 35.1
MAT 112S using an ionization voltage of 60 eV. It was 1.66 (6) 3.8 4.2 3.7 19.8 20.2
equipped with an AMD Intectra DP-10 data system with a 1.68 (7) 50.2 55.5 47.6 5.9 3.7
Wiley library. For sterols and triterpene alcohols, the deter- 1.84 (8) tr tr tr 9.8 8.2
mination was carried out on the GC with an OV-17 glass 1.95 (9) 7.8 6.9 7.5 tr -
2.17 (10) 8.9 5.2 9.8 tr 1.2
capillary column (30 m × 0.3 mm i.d). Relative retention times
2.19 (11) 5.6 4.8 7.8 1.5 2.3
(RRT) were expressed as silanated cholesterol ether (1.00). The
unidentified peaks 7.6 5.2 8.0 5.6 6.7
RRT values of authentic samples of sterols and triterpene
alcohols are given in Table 3. a Components add up to 100% in each column. -, below
Table 5. Composition of the Triterpene Alcohols of Five Table 7. Distribution of Hydrocarbons in Five
Underutilized Tropical Seed Oils Underutilized Tropical Seed Oils
plant speciesa plant speciesa
RRT TLO ADB CME ATF MNT peak TLO ADB CME ATF MNT
1.30 (12) 1.9 1.2 3.6 8.2 7.4 1 - - - 2.2 10.3
1.33 (13) 3.7 5.2 6.2 tr tr 2 - - - - 1.8
1.50 (14) 2.7 - tr 3.7 3.2 3 1.4 1.7 - - 2.8
1.58 (15) tr tr tr 7.5 6.2 4 0.6 0.7 - - 2.8
1.66 (16) 25.1 23.4 20.7 9.6 8.8 5 - - 2.3 - 2.2
1.72 (17) 7.7 6.8 5.8 3.0 3.4 6 0.6 0.6 - 1.4 0.7
1.77 (18) 22.5 22.3 23.7 - - 7 - 4.3 7.0 6.8 4.7
1.79 (19) 2.1 1.2 1.5 1.1 2.5 8 1.0 1.1 1.4 2.9 1.5
1.87 (20) 5.1 6.1 5.8 36.2 38.6 9 2.5 2.6 2.3 3.7 11.9
1.89 (21) 3.2 5.8 4.2 9.5 8.2 10 3.5 3.5 4.2 4.1 2.9
1.90 (22) 1.0 - - - - 11 15.3 16.6 18.0 6.8 5.1
1.96 (23) 11.5 10.6 12.7 3.2 6.0 12 - 3.5 4.2 4.1 2.9
2.10 (24) 5.8 10.3 8.9 10.4 6.5 13 6.2 5.6 5.6 5.9 4.7
2.40 (25) 0.2 0.3 0.6 1.5 2.3 14 17.0 8.5 6.5 23.3 5.1
2.52 (26) tr tr tr 0.2 0.7 15 - 5.4 3.6 5.9 6.7
unidentified peaks 7.5 6.8 6.3 5.9 6.2 16 7.8 6.9 5.6 5.9 5.9
a Components add up to 100% in each column; tr, trace
17 7.4 4.8 4.5 4.1 5.1
18 12.3 16.0 7.9 6.8 6.1
concentration; -, not detectable. 19 6.5 5.8 7.1 5.2 4.7
20 5.8 5.3 6.8 3.7 3.7
Table 6. Hydrocarbon Fractions Identified in Five
21 5.1 3.2 4.9 3.7 3.5
Underutilized Tropical Seed Oils
22 3.1 1.7 3.4 2.1 1.9
code hydrocarbon code hydrocarbon 23 2.5 1.1 3.3 1.4 1.5
24 1.4 1.1 1.4 - 1.0
1 R-piene 13 pentacosane (C25H52)
2 camphene 14 hexacosane (C26H54) a Components add up to 100% in each column; -, not detectable.
3 myrcene 15 octadecanoic acid
4 p-cymene butylamide
5 octadecane (C18H38) 16 heptacosane (C27H56)
6 nonadecane (C19H40) 17 octacosane (C28H58)
7 ethyl ester of palmitic acid 18 squalene
8 heneicosane (C21H42) 19 nonacosane (C29H60)
9 docosane (C22H46) 20 triacotane (C30H62)
10 tricosane (C23H48) 21 hentriacotane (C31H64)
11 tetracosane (C24H50) 22 dotriacotane (C32H66)
12 hexadecanoic acid 23 tritriacotane (C33H68)
butylamide 24 tetraacotane (C34H70) Figure 1. Structure of isomultiflorenol.
technical support. We thank L. Lajide and O. Ekundayo Esuoso, K. O.; Odetokun, S. M. Proximate chemical composi-
for their advice. tion and possible industrial utilisation of Blighia sapida
seeds and seed oils. Riv. Ital. Sostanze Grasse 1995, 72,
311-313.
LITERATURE CITED
Esuoso, K. O.; Lutz, H.; Kutubuddin, M.; Bayer, E. Chemical
Akihisa, T.; Ghosh, P.; Thakur, S.; Rosenstem, F. U.; Matsu- composition and potentials of some underutilised tropical
moto, T. Sterol composition of seeds and mature plants of biomass I. Fluted pumpkin: Telfairia occidentalis. Food
the family, Cucurbitaceae. J. Am. Oil Chem. Soc. 1986, 63, Chem. 1998, 61, 487-492.
653-658. Heftmann, E. Recent Advances in Phytochemistry; Runecklen-
Akihisa, T.; Inada, Y.; Ghosh, P.; Thakur, S.; Rosenstem, F. sad, V., Steelink, C., Eds.; Appleton Century-crafts: New
U.; Tamura, T.; Matsumoto, T. Composition of the triterpene York, 1970; Vol. 3.
alcohols of seeds and mature plants of the family, Cucur- Jalad, S. D.; Wiederhopf, R. M.; Cole, J. R. Cytotoxic agents
bitaceae. J. Am. Oil Chem. Soc. 1988, 65, 607-610. from Bursera klugii. J. Pharm. Sci. 1977, 77, 889-890.
Andriamanantena, R. W.; Artaud, J.; Gaydou, E. M.; Iatrides, Kamal-Eldin, A.; Yousif, G.; Iskander, G. M.; Appelquist, L.
M. C.; Chevalier, J. L. Fatty acid and sterol compositions A. Seed lipids of Sesamum indicum L. and related wild
of Malagasy Tamarind kernel oils. J. Am. Oil Chem. Soc. species in Sudan I: Fatty acids and triacylglycerols. Fat Sci.
1983, 60, 1318-1321. Technol. 1992, 94, 254-259.
Bastic, M.; Bastic, M.; Javanovic, J. A.; Spittler, G. Sterols of Kupchan, S. M.; Meshulam, H.; Sneden, A. T. New cucurbitans
pumpkin seed oil. J. Am. Oil. Chem. Soc. 1977, 54, 525- from Phomiun tenax and Marah organus. Phytochemistry
527. 1978, 17, 767-769.
Capella, P.; Iversion, L. J.; Crimele, M.; Jacini, G. Studies on Nakatani, M.; James, J. C.; Nakanishi, K. Isolation and
the distribution of hydrocarbons in some vegetable oils. Riv. structures of trichilins antifeedants against Southern ar-
Ital. Sostanze Grasse 1963, 40, 603-608. myworms. J. Am. Oil Chem. Soc. 1981, 103, 1228-1230.
Dreyer, D. L.; Trousdale, E. K. Cucurbitacins in Purshia Oboh, F. J.; Oderinde, R. A. Analysis of the pulp and pulp oil
tridentata. Phytochemistry 1978, 17, 325-326. of fat of tucum, Astrocaryum vulgare. Food Chem. 1988, 30,
Ekundayo, O.; Hammerschmidt, F. J. Constituents of the 277-287.
essential oils of Monodora myristica seeds. Fitoterapia 1988, Vasconcellos, J. A.; Berry, J. W.; Werber, C. W.; Beems, W.
59, 52-54. P.; Scheerer, J. C. Properties of Cucurbita foetidissima seed
Esuoso, K. O. Nutritive properties of monkey pod (Samanea oil. J. Am. Oil Chem. Soc. 1980, 57, 310-313.
saman). Riv. Ital. Sostanze Grasse 1996, 72, 34-38.
Esuoso, K. O.; Bayer, E. Chemical composition and potentials Received for review July 30, 1999. Revised manuscript received
of some underutilised tropical biomass II. Andenopus brevi- November 8, 1999. Accepted November 16, 1999.
florus and Cucumeropsis edulis. Riv. Ital. Sostanze Grasse
1998, 75, 191-195. JF9908493