Limonoids: Pesticide to anticancer applications from secondary metabolites
of the Rutaceae and Meliaceae
David Bagge
Colorado State University
Fort Collins, Colorado
dbagge@CVMBS.Colostate.edu
Abstract
Limonoids are described as modified triterpenes with or derived from a
precursor with a 4,4,8-trimethyl-17-furanylsteroid skeleton. Over 300 limonoids
have been isolated to date. Past work has established a wide range of
biological activities for these compounds including insect anitfeedant and
growth inhibiting characteristics. Although best known for their insecticidal
properties a variety of medicinal effects in animals and humans has been
illustrated including some anticarcinogenic effects on in-vitro human cancer
cell lines and test animals. Other limonoid properties include antifungal,
bactericidal, and antiviral effects.
The insecticidal aspect of these compounds gained economic importance with
the application of the highly effective limonoid, azadirachtin. Current
literature for the last two years is devoted to the identification of Rutaceae
and Meliaceae species containing limonoids. Extraction and isolation methods
make up a bulk of recent publications with eventual study of the limonoids as to
biological activity exhibited toward insect species. Lethal concentrations to
50% of test insect speicies (LC50) of most limonoids studied fall in the range
of 50 parts per million (ppm) or lower, with some exhibiting LC50 s as low as
.625 ppm, depending on the insect studied. The effect of ring structure and
chemical oxidation state parameters is a focus of why limonoids exhibit
activity against insect herbivores. The variety of additional biological
properties exhibited by limonoids has also contributed to their interest to the
scientific community. The future course of their study focus may well evolve
into their use against cancer, bacteria, viruses and fungi.
Limonoids are secondary metabolites produced in plants found in the order
Rutales. Within this order, limonoids are most often found in the family
Meliaceae and less frequently in the families Rutaceae and Cneoraceae. Over 300
limonoids had been identified by 1992, with many more being extracted and
isolated from citrus species in the past five years.
Limonoids are described as modified triterpenes, having a 4,4,8 trimethyl
- 17 furanyl steroid skeleton. Arrangements of subgroups and ring structures
within this basic building block provide a host of characteristics that have
generated interest in this plant product. These characteristics include
insecticidal, insect growth regulation, insect antifeedant, and medicinal
effects to animals and humans such as antibacterial, viral, and antifungal
properties. Of recent great interest, limonoid's possible anticarcinogenic
properties are being explored.
Further interest in limonoids is generated by their economic impact on the
citrus fruit and juice industry. The ability of some limonoids to produce an
extremely bitter taste in these products has provided much impetus to the study
of their structural makeup and characteristics.
Limonoids Verses Insects
Limonoids appear to be a plant chemical response to insect herbivory.
Studies in recent years have revealed insecticidal effects of many different
limonoids. The specific effects studied include growth inhibition, feeding
inhibition, molt inhibition, and insect growth regulation. Most studies have
focused on the insect orders of Coleoptera, Diptera,
Heteroptera, Lepidoptera, and Orthoptera. Azadirachtin (an Asian limonoid) or
azadirachtin containing extracts have been shown to affect over 200 species of
insects and mites (1, 2).
Unfortunately, a direct understanding of limonoid properties is hampered
by many factors. Investigators seldom utilize the same bioassay species and
many times differences in larval stages tested may make comparisons invalid (3).
In addition, the view that limonoids are primarily antifeedant chemicals
has contributed to a majority of studies that fail to detect non-behavioral
effects such as toxicity and growth regulation (4).
Even with some of these shortcomings, studies have shown some basic
properties that the chemical structure of a particular limonoid imparts on
insects. First, an understanding of the basic building structure of limonoids is
necessary. Euphane and tirucallane are two very similar chemical precursors that
give rise to ten different grouping of limonoids. These precursor's structure
is illustrated in Figure 1.
Figure 1. Euphane: Basic structure of a limonoid precursor.
In most limonoids the aliphatic straight carbon chain at the top is changed to a
euphol group.
Figure 2. Euphol structure found on the furan ring of most limonoids.
Limonoids are usually grouped according to changes they undergo in one or
more of their four-ring structures. The rings are designated as A, B, C, or D
as shown in Figure 1. Four limonoid skeletal structures are illustrated in
the next figures to show how ring structure changes in groups.
Azadirachtin is a limonoid that has been commercially used as a pesticide.
(Figure 3) It is classified in the C-seco group.
Figure 3. Azadirachtin skeletal structure, illustrating its specific change
in the C ring. Side groups are not shown but oxygen incorporated into ring
structures are illustrated. (True for all subsequent figures.)
Azadirachtin has an open C ring, although some other members of the C-seco
group's C rings are closed, with an oxygen added to the other components of the
ring. This group produces moult inhibition, antifeedant activity and growth
inhibition in studied insects at concentrations of 1 part per million (ppm) or
less(5).
Cedrelanolide is a B-seco limonoide extracted from stem bark of Cedrela
salvadorensis, a small tree of the dry Pacific slope of Mexico(6 ).
(Figure 4.)
Figure 4. Cedrelanolide, illustrating change in the B ring.
Since this is a recently isolated limonoid, no studies as to its effects on
insects have been noted in the literature. Other B-seco limonoids such as
Toonacilin exhibit feeding inhibition in the 2000 ppm range and probably would
not be of commercial interest (4).
Limonin is an A,D seco limonoid of great commercial concern due to its property
of imparting bitternes in some citrus juices. This property caused it to be the
first limonoid extracted and isolated for study. Both the A and D rings of these
limonoids have an oxygen added or substituted into the ring structures. Limonin
inhibits growth to 50 percent of insect species studied (EC50) at
concentrations of 700 to 1000 ppm. This does not make it desirable as a
commercial insecticide. Another A,D seco limonoid is nomilin, with EC50s in the
range of 70 to 100 ppm. Some members of this group may show commercial promise
as an insecticide. It may be that these limonoids function mainly as anti
feedant chemicals. They are found in very high concentrations (> 1000 ppm) in
many citrus plants(Figure 5).
Figure 5. Limonin, illustrating change in the A and D ring structure.
The final figure illustrates Toosendanin, another limonoid found in the
Far East. It is described an intact apoeuphol limonoid, with no ring
alteration. Like Azadirachtin, it has been extracted and used as a crop
protectant. It is slightly less potent though, showing feeding and growth
inhibition to studied insect species at levels of 200 to 400 ppm (Figure 6).
Figure 6. Toosendanin, an intact apoeuphol limonoid.
There are about 10 groupings of limonoids based on how the specific rings
are structured and elemental or chemical groups that are incoporated into or
onto them. These figures serve to illustate how the groupings are derived.
It appears that the addition of oxygen incorporation into ring structures
and side groups to the cyclohexane ring structures contribute to the ability of
these secondary plant compounds to damage insects. One such oxygen arrangement
found on many of the limonoid groups is an epoxide. This addition of an oxygen
to two carbons formerly possessing a double bond between them is a toxic
metabolite often bioactivated by enzymes in humans (7). These oxygen
additions are often very reactive and often cause problems by adducting to DNA
or binding enzymes. They may play the same role in the metabolism of insects.
One point reported in a few studies is that bioassyas to date have used
oligophagous and polyphagous species of insects that rarely or never feed on
limonoid containing plants. Limonoid insecticides probably are not good for
control on citrus crops but may prove to be economically feasible for others
(1).
The C-seco limonoids are found only in the two related genera Azadirachta
and Melia which suggest tha they are relatively specialized compounds which
evolved concurrently with the separation of the Melieae as a distinct tribe.
This separation has led to speculation on the selection pressure from insect
herbivores as a driving force in the evolution of the various limonoid groups.
At this point in time it is not possible to draw any conclusions regarding
limonoid evolution because of the lack of specific studies on structural
activity. All bioassays to date (1995) have used polyphagous or oligophagous
insects that rarely or never feed on limonoid-containing hosts. The role of
limonoids in a speculated coevolutionary relationship between plants and insects
would have to be examined in the context of the adapted insect species feeding
on limonoid containing plants (4).
Limonoids as Bitter Substances
The citrus juice industries of the world are greatly influenced by the
characteristics of some limonoids. Most citrus fruits do not taste bitter if
eaten fresh or if freshly squeesed juice is consumed. However, within a few
hours after juicing at room temperature, or overnight if stored in a
refrigerator, the juice extractd from some winter citrus variteties become
bitter. Those varieties include navel orange, grapefruit, Natsudaidai, Iyo
hybrids, pummelo hybrids and others. (8) This gradual development of
bitterness, or delayed bitterness, in these citrus juices is caused by the
formation of limonin, from a tastless precursor. The mechanism of delayed
bitterness was not fully understood until 1968, when limonin was isolated. The
economic importance of the bitterness in juice products provided the impetus for
the structural description of limonin, an impressive achievement for chemical
analysis at that time. (9) The precursor was finally identified a year later as
limonate A-ring lactone, which is present in the carpellary membrane and albedo
tissue of fruits. Intact fruit normally contains only the precursor. After
juice is expresessed from citrus fruits they gradually develop a bitter taste.
The causative factor was shown to be limonin.
After juice is extracted, this precursor is gradually converted to
limonin. The reaction proceeds under acidic conditions below pH 6.5 and is
accelerated by the enzyme limonin D-ring lactone hydrolase. (10) The delayed
bitterness lowers the quality and value of commercial juices and has significant
negative economic impact to the citrus industry. Unusual weather and harvesting
conditions which cause disruption of fruit tissues, such as freezing or
mechanical damage, can promote the acidic pH and enzyme activity in the fruit
tissues and the conversion of the precursor to limonin. (11) Fresh citrus
juices are usually not bitter, but after a short time, they develop an
unpleasant taste. Various lines of evidence showed that a non-bitter precursor
was converted to limonin following disruption of fruit tissues.
Limonin is exceedingly bitter and is shown detectable by human test
subjects at concentrations ranging from .075 to 5 ppm. (8 ) Other bitter
tasting limonoids are nomilin, and ichangin, although they are found is much
lower concentrations than limonin in the fruits. Debittering processes have
recently been described using UDP-D-limonin glucosyltransferase. This enzyme
catalyses the glucosylation of both limonin and nomilin. It is isolated and
purified from the albedo tissues of naval orange cultivars. The synthetic
manufacture is being looked at for and economical means of juice flavor
enhancement. As with any new process, tests on product stability and health
effects will have to undertaken to assure no long term adverse effects with the
addition of this enzyme. (12)
Limonoids and Treatments for Human Illnesses
Extracts of many citrus species are used traditionally in the herbal
medicines of the Far East. The fruits and bark of the Chinaberry (Melia
azedirach) have been used as a treatment for a variety of ailments in small
doses, as it can be toxic to humans. (1)
Medicinal properties of Azadirachta indica (neem) are also recognized
historically. Neem, a derivative of these plants has been used to treat blood
disorders, hepatitis, cancer, ulcers, constipation, syphilis, snake bite and a
host of other ailments. (13) They appear to work well as topical formulations,
finding success in the treatment of fungal infections and parasiticides. Many
of these activities are being substantiated by current research.
Modern applications include the use of the limonoid components neem,
nimbidinic acid, and nimbolide. These compounds have diuretic effects in test
animals. (14) Sodium nimidinate is speculated to be the causal agent of neem
oil spermicidal activity. Nimbin has also been shown to protect against the
ulcerative activity of aspirin and serotonin in rats and guinea pigs. (15)
Of special interest to countries in tropical locations is the antimalarial
activity attributed to tropical meliaceae extracts and gendunin derivatives.
Nibolide is shown to inhibit the growth, in vitro, of Plasmodium berghi, which
causes malaria in humans. Unfortunately, it has no in vivo inhibition in mice.
Gendunin is also a limonoid found in A. indica and has been found to inhibit
Plasmodium falciparum, the most active parasite for the cause of malaria
worldwide. The antimalaria activity of these limonoids has been linked to some
of the reactive sites in their A rings. (16)
Nimbidin applied as .1, 1.0, and 5.0 percent solutions in dilute ethanol
inhibited the growth of many bacterial and fungal species. Just the same,
species such as Eschericia coli, many citrobacter species, and Klebsiella
pneumoniae showed no effects of nimbidin presence. It must be noted also that
the inhibition is occurring at very high concentrations of nimbidin, 5.0
percent equals 50,000 ppm, and enormous dose for any pharmacological compound
used for treatment.
Limonoid Anticarcinogenis and AntiMutagenic Activity.
Some of the most exciting applications of limonoids and compounds derived
from them are their use in the treatments of specific cancers. Limonin,
nomilin, 12,hydroxyamdorastatin, and isofraxinellone are limonoids or their
derivatives that have been shown successful in treatments with in vitro
bioassays on human tumor cell lines. (17)
Limonin and nomilin were described earlier as being bitter principals for
citrus fruits. Both limonoids have been found to induce increased activity of
the detoxifying enzyme glutathione-S- transferase. The increased enzyme
activity was correlated with the ability of these compounds to inhibit
chemically induced carcinogenisis in laboratory animals. Administration of
nomilin by gavage to a specified strain (ICR/Ha) of mice reduced the incidence
and number of forestomach tumors per mouse induced by benzo [a] pyrene (BP), a
potent epoxide former. (1).
Addition of nomilin to the diet at various concentrations inhibited BP
induced mice lung tumor formation. This was attributed to the limonoid's
inhibition of the formation of BP-DNA adducts in the lung. (18)
Topical application of limonoids was found to inhibit both the initiation
and promotion phases of carcinogenisis in the skin of [SENCAR] mice. Nomilin
appeared to be more effective during initiation stage induced carcinomas while
limonin was more potent as an inhibitor during the promotion phase of the
carcinogenisis. (17) These and other findings (19, 20) suggest citrus limonoids
may be useful as cancer chemo-preventative agents.
Recent studies into induced oral cancers in hamsters suggests that limonin
can act as a capture chemical, intercepting compounds such as benzo [a] pyrene
and other mutagens before the formation of adducts to cell macromolecules.
Since oral cancers are on the rise in human populations, the treatment by an
easily isolated and obtained compound would be of great interest to the oncology
profession.
Conclusions
The insecticidal properties of many limonoids, especially C-seco, and many
more widely distributed intact apo-euphol skeleton, 14,15 epoxide limonoids, are
driving many recent studies in the extraction and isolation of these compounds
from new sources. It may be though, that continued elucidation of limonoid
compounds would be developed by their activity against organisms other than
insects. Indications of the antifungal, bacteriacidal, protisticidal and
antiviral characteristics suggest a broader role for these compounds.
As an economic concern, the neutralization of the chemical compounds that
cause citrus bitterness in fruits and juices is a prime concern of this produce
industry. The increase in the purification process of UPD-D -glucose limonoid
glucosyltransferase may provide a feasible method for future debittering
methods.
As a health conscious society with increasing concern and desire for
effective cancer treatments, the limonoids are extremely attractive. With over
300 similar compounds, the bioassay of known limonoids for their anticancer and
antimutagenic activities will encourage much more investigation. Like many of
their secondary plant metabolite cousins, limonoids appear to be a source of
countless possible resources that can benefit the human race.
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