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Ethnobotanical Leaflets 14:797-806. 2010. Epicuticular Wax and
Volatiles of Kigelia
pinnata
Leaf Extract Atolani
Olubunmi1* and Olatunji A. Gabriel2 1Department
of Chemical Sciences, Redeemer’s University, PMB 2011, 2Department of Chemistry, University of E-mail: atolanio@run.edu.ng Issued: July 1 2010 Abstract The fractions of volatile compounds in plants are essential in defining
the sensory impact of an aromatic smell, as well as being of some medicinal
importance. Epicuticular wax in plants also
determine its susceptibility to
disease, and herbivore, desiccation and ultraviolet radiation damage. The epicuticular wax consisting of hydrocarbons and some
volatile compounds of the leaves of Kigelia pinnata (Kigelia africana; family Bignoniaceae),
a multimedicinal plant, was extracted, isolated and
analyzed qualitatively and quantitatively for the various chemical
compositions using GC (equipped with a flame thermionic detector -GC/FTD) and
GC-MS (“GCMS-QP2010 Plus, which employs a dual inlet Turbo Molecular Pump
(TMP) that provides increased sensitivity and column flow capacity for
improved performance and confident results. The evaluation is useful for the
future comprehensive analysis of the constituents responsible for the various
documented medicinal uses of the leaf. The extracted constituent revealed
twelve compounds with the major ones identified as n-hentriacontane
(55.40%): a probable anti-tumour compound;
1-tricosene, (18.45%); 11- (2,2-dimethylpropyl) heneicosane,
(9.66%); 2,6,10-trimethyldodecane, (4.43%); pentafluoroheptadecyl
ester, (4.40%); 2-ethylhexyloctadecyl sulfurous acid ester, (3.05%); heneicosane, (1.61%); and hexyloctyl
sulphurous acid ester, (1.42%). Other compounds are
recorded in minute quantities. The major component, identified as hentriacontane,
a C-31 saturated hydrocarbon apparently is responsible for the plant
susceptibility to diseases, protection against UV radiation and for the antitumour and antioxidative
potential of the plant. This will give credence to the traditional use of the
plant as an anti-inflammatory and anticancer. The ability of the plant to act
as anti-sun burn may be due to the presence of the wax. Key words; Kigelia pinnata, Kigelia africana, Epicuticular wax, Bignoniaceae, volatiles, GC-MS, hentriacotane. Introduction The pharmaceutical, cosmetic and food industries are constantly being faced with the challenge of identification, isolation and characterization of volatile compounds of medicinal importance in plant materials. Kigelia pinnnata (Jacq) DC, also known as Kigelia africana (Lam) Benth and sausage tree on account of its large fruits, is a member of the family Bignoniaceae. It is a multipurpose medicinal plant with great potentials to be developed as drug by pharmaceutical industries (Sangita et al., 2009, Olatunji and Atolani, 2009). The traditional uses of the stem, bark, root, leaves and fruit are enormous. Studies that have been reported on the leaf of this plant are rather limited. The plant’s crude extract is used as antiageing and antiburn in cosmetic such as cream. The plant is rich in volatile constituents. Phytochemical studies on the stem bark revealed the presence of Kigelinone and isopinnatal (Akunyili and Houghton, 1993), specioside, verminoside and minecoside (Picerno et al., 2005; Neelam et al., 2006) and phenylpropanoid (Gauda et al., 2006). The root has been reported to possess isopinnatal, kigelinol and isokigelinol (Moideen et al., 1999) and p-coumaric acid (Binutu et al., 1997). Flavonoids and irridoids have been isolated from its leaves (Gouda et al., 2003). The
anti-diarrhoea activity of the aqueous leaves extract has been confirmed (Akah, 1996). The ethanolic
extract of the stem bark was examined to show strong analgesic and
anti-inflammatory activities by inhibiting the synthesis of prostaglandins
and other inflammatory mediators, (Owolabi and Omogbai, 2007). The stem bark extract have been reported
for their cytotoxicity activities (Houghton et al., 1994), Central Nervous System
Stimulation (Owolabi et al, 2008), antifungal activity (Jain and Belsare,
2009) and anti-trypanosomal activity against trypanosome brucel brucei in vitro (Moideen
et al., 1999), while the root
extract have been reported for its positive anti- uterus cancer against
malignant melanoma (Skin cancer) (Houghton et al., 1994 and Msouthi and Mangombo, 1983). Epicuticular waxes are
partially saturated lipid compounds located on the above-ground portions of
land plant (Post-Beittenmiller, 1996). The chemical
composition of waxes varies by species: in maize, the epicuticular
wax is composed of fatty acyl chains that are later
converted to alcohols, esters and aldehydes and a
small proportion of alkanes (Bianchi et al., 1985 and 1989) Flavonoids, fatty acid esters could also be found in epicuticular waxes (Schmutz et al., 1994; Whitaker et al., 2001). Epicuticular
wax have been extracted, purified by column and thin layer chromatography (Kolattukudy, 1965 and 1966) and reported to determine
plant susceptibility to diseases, herbivore, desiccation (Juliana and Silvana, 2006) and ultraviolent radiation protectant (Lacy et
al., 2003) To
the best of our knowledge, very little has been reported on the phytochemistry and medicinal use of the leaves of K. pinnata, despite its many
traditional applications. Hence it is important to demystify the compositions
of the components of the leave. This constitutes the main objective of
this study. Materials and Method All
the solvents used were obtained from the Department of Chemistry, Extraction Kigelia pinnata leaves were dried at room temperature. The
air-dried plant material was blended into powder at a low temperature. 240g
of the powdered plant material was extracted exhaustively with cold hexane
for six days. The extract was combined, concentrated in vacuum and 3.06g of
brownish-green syrup was obtained. Isolation of volatiles The
syrup was applied to a column chromatography (CC) packed with Silica gel and
eluted with hexane. Eleven fractions were collected, examined (talc) and
combined as appropriate. The major combined fraction was subjected to a
silica gel flash chromatography. Six yellow fractions with same Rf (silica gel) were obtained, combined and
evaporated to dryness. This was coded KPLH/CC/FC/0.5. The chromatoplate
was sprayed with vanillin spray reagent for detection of other spots. Separation of Component The
volatile fraction (KPLH/CC/FC/0.5) was analysed by gas chromatography (GC)
using a GC 2010 gas chromatography (Shimadzu, Japan) equipped with a flame
thermionic detector -GC/FTD
and an electronic high pressure control injector. The flow of the carrier gas
(He at 100.2 kpa) 1.61mL/min; linear velocity:
46.3cm/sec; total flow: 6.2mL/min; purge flow: 3.0mL/min. The split ratio is
1:0. The analysis was performed using the following program; oven kept
isothermally at 60oC for 6 min, increased from 60oC to
220c at a rate of 25oC/min and kept isothermally at 220oc
during 5min. It was increased to 280oC at a rate of 5oC/min
for 20min; Equilibrium time, 3min. Total ion chromatogram (TIC), 1.0 GC-MS Analysis Gas
Chromatography-Mass Spectroscopy, GC-MS System; GCMS - QP2010 PLUS (SHIMADZU
JAPAN) interfaced with a finigan MAT ion trap
detector ion source Temp., 200oC; interface Temp, 250oC;
solvent cut time, 2.50min; relative detector gain mode, ACQ mode; Scan: start
time - end time; 3.00min - 46.00min; event time, 0.50 sec; Scan Speed, 1428.
Identification of the volatile components was carried out using the peak
enrichment technique of reference compounds and as final confirmation of the
peak identification by GC-MS, comparing their spectra with those of the NIST
commercial library mass spectra. FT-IR analysis The
infrared spectrum of the volatile was recorded on a Shimadzu (8400S) Fourier
Transform-Infrared Spectroscopy (FTIR) Spectrum Spectrophotometer using KBr pellet. Results and Discussion A
total of twelve compounds were detected in the fraction (KPLH/CC/FC/0.5) of Kigelia pinnata examined.
The compounds identified, the retention times, peak areas, percentage yields
and base peaks are shown in Table 1. The IR spectrum of the isolated epicuticular wax showed υmax (cm-1, KBr)
3479 – 3282, (OH); 2955 – 2850, (CH2, CH3); 1650,
(C=O); 1462 and 1377, (CH2, CH3). About 91% of the
total isolate is hydrocarbon. Hentriacontane (55%), a
saturated hydrocarbon, the major compound identified has been isolated from Scabiosa comosa (Dargaeva
and Brutko,
1976). It is reported to be responsible for its uptake in the soil by
plant (Éric, 1995) and shown to be involved
with stimulation of fungal spore germination (Dauri and José, 1995). Hentriacontane has
also been isolated from spinach leaves, and discovered to be unsaponifiable (Edith and Ida 1929) and shown to have
possible anti-tumour activity (Takahashi et al., 1995). Methyl 12- Methyl tetradecanoate, a lipid of biological significant was
also identified in a microscale. It has been
identified in trace amount in Cryobacterium
psychrophilum (Ken-Ichiro et al.
1997), Xylella fastidiosa plant (Ana Valéria et al.,
2007), and Corynebacterium
sepedonicum
plant (Paul et al., 1988). It was
reported for its inhibition capacity on the development of corneal
angiogenesis, which is responsible for blindness and other infections (Cole et al., 2007). The three main non-hydrocarbons obtained are
2-ethylhexyl-octadecyl ester (3.05%), heptadecyl
ester (pentafluoropropionic) (4.40%) and hexyloctyl ester (1.42%). Tetradecanoic
acid (12-methyltetradecanoate) was obtained in a low quantity (0.29%). Hexadecanoic acid has earlier been reported as a
component in alcohol extract of the leave of Kigelia pinnata (Grace et al., 2002). The components reported
in this work have not been reported in literature before. In
conclusion, twelve volatiles were identified and characterised by the
GC/GCMS. The major component, identified was hentriacontane,
a C-31 saturated hydrocarbon which might be apparently responsible for the
plant susceptibility to diseases, herbivore, desiccation, protection against
UV radiation and for the antitumour and antioxidative potentials of the plant. This will give
credence to the traditional use of the plant as an anti-inflammatory and
anticancer. The ability of the plant to act as anti-sun burn when used in
cream may be partly due to the presence of the wax. We hereby report
that the major paraffin in the epicuticular wax of Kigelia pinnata
leave is n-hentriacontane, an n-C31 hydrocarbon
(Kolattukudy, 1965 and 1966). ACKNOWLEDGEMENTS We
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KRI: Kovats Retention index |