Insect Control: Biological and Synthetic Agents - Index of

188 5: Azadirachtin, a Natural Product in Insect Control
groups. The molecule contains an acetate, a tiglate
ester, two methyl esters, a secondary and a tertiary
alcohol, an epoxide, a vinyl ether, which is part of
an acetal, and a hemiacetal. This overabundance
of reactive groups makes its chemistry very difficult
(Ley et al., 1993). Just how these contribute to its
insecticidal properties is not yet understood. Another
locust antifeedant called meliantriol of lower activity
than azadirachtin was announced shortly before the
isolation of azadirachtin (Lavie et al., 1967), but its
isolation has never been repeated.
Structural studies on azadirachtin began immediately
after its isolation, but because of its complexity
and the sensitivity of some of the functional groups,
progress was difficult. The structure was finally
solved 18 years after isolation by a combination of
modern nuclear magnetic resonance (NMR) techniques
and X-ray crystallography (Bilton et al., 1987;
Kraus et al., 1987; Turner et al., 1987). Synthesis of
azadirachtin presents a great challenge to organic
chemists. Great progress has been made towards it
by the groups of Ley (Durand-Reville et al., 2002)
and others (Nicolaou et al., 2002; Fukuzaki et al.,
2002), but as yet it has not been completed.
5.3.1. Neem Limonoids
Largely because of the interest in azadirachtin, the
triterpenoids of neem have been studied intensively.
About 150 such compounds have now been described
(Akhila and Rani, 1999), most of them found in very
small quantities in various parts of the tree. Only
about one-third of them have been tested for biological
activity, and none has shown greater activity than
the azadirachtin group.
Unfortunately many writers speak of ‘‘neem’’ as
if that were a single commodity. The different parts
of the neem tree all have different properties and contain
different substances. The lack of more accurate
descriptionandthefrequentlackofanalyticaldata
on what is contained in seed extracts have reduced
the value of some of the published work on such extracts.
The seeds are the only practical source of azadirachtin
and its group of compounds. Any of this
substance in other parts of the tree is in marginally
small concentration.
5.3.2. Isolation of Azadirachtin
The triterpenoid present in greatest quantity in
the seeds is usually salannin (Figure 1), a simpler
triterpenoid with only weak activity as a feeding
deterrent. Azadirachtin, representing 0.1–1.0%
(mean 0.6%) of the weight of the seed kernels is
next in quantity. The other compounds of azadirachtin-like
structure and biological properties occur
in progressively smaller amounts. Chemically, they
divide into three structural types (Figure 2): the
azadirachtins, with a hemiacetal group at carbon
atom number 11, the azadirachtols, without the
hemiacetal, and the meliacarpins, in which the
methoxycarbonyl ester at C29 is replaced by methyl.
Not all compounds found fit easily into these
types and new trace constituents continue to be
found (e.g., Luo et al., 1999; Malathi et al., 2003).
Rearrrangement products, known as azadirachtinins
(Figure 3), of much lower biological activity,
are found in the seeds and are also formed during
isolation of the limonoids. The names azadirachtin
A, azadirachtin B, etc., are sometimes used. These
are incorrect names. For the correct chemical names
for all these triterpenoids and limonoids, see Kraus
(2002). Also present in lesser quantities are nimbin,
3-desacetylnimbin, and 6-desacetylsalannin (Johnson
et al., 1996) (Figure 1), simpler limonoids of lesser
activity and interest. Compounds of similar structure
are found in the related Melia genus. For example,
Persian lilac or chinaberry, Melia azedarach,contains
meliacarpins (Figure 2), but the seeds are extremely
hard and their extracts are toxic to mammals.
A number of isolation procedures have been
described, but all require extraction with solvent
from the ground seeds, followed by solvent partition
to separate the more polar triterpenoids from
the oil. After that, methods differ more, but all
require some form of chromatography to separate
the individual compounds, either gravity column
chromatography (Johnson and Morgan, 1997a),
flash columns (Jarvis et al., 1999), or preparative
high-performance liquid chromatography (HPLC)
(Lee and Klocke, 1987; Govindachari et al., 1990,
1996). Extraction by supercritical carbon dioxide
has been examined, but is not as efficient as solvent
extraction (Johnson and Morgan, 1997b;
Ambrosino et al., 1999). There is a lot of current
interest in microwave-assisted chemistry, and a
microwave-assisted extraction of seeds has been
described (Dai et al., 2001).
5.3.3. Analysis
The number of triterpenoids in neem seeds and their
similarity in physical properties makes analysis for
the important pesticidal compounds both important
and difficult. The standard method in use for some
time is reverse-phase HPLC with ultraviolet (UV)
absorption at short wavelength (214–218 nm) (e.g.,
Deota et al., 2000). Normal-phase supercritical fluid
chromatography offers advantages where the equipment
is available (Johnson and Morgan, 1997a).
A colorimetric method for the whole group of triterpenoids,
using vanillin–sulfuric acid has been